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Photoaffinity Labeling of Pentameric Ligand-Gated Ion Channels: A Proteomic Approach to Identify Allosteric Modulator Binding Sites

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Neuroproteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1598))

Abstract

Photoaffinity labeling techniques have been used for decades to identify drug binding sites and to study the structural biology of allosteric transitions in transmembrane proteins including pentameric ligand-gated ion channels (pLGIC). In a typical photoaffinity labeling experiment, to identify drug binding sites, UV light is used to introduce a covalent bond between a photoreactive ligand (which upon irradiation at the appropriate wavelength converts to a reactive intermediate) and amino acid residues that lie within its binding site. Then protein chemistry and peptide microsequencing techniques are used to identify these amino acids within the protein primary sequence. These amino acid residues are located within homology models of the receptor to identify the binding site of the photoreactive probe. Molecular modeling techniques are then used to model the binding of the photoreactive probe within the binding site using docking protocols. Photoaffinity labeling directly identifies amino acids that contribute to drug binding sites regardless of their location within the protein structure and distinguishes them from amino acids that are only involved in the transduction of the conformational changes mediated by the drug, but may not be part of its binding site (such as those identified by mutational studies). Major limitations of photoaffinity labeling include the availability of photoreactive ligands that faithfully mimic the properties of the parent molecule and protein preparations that supply large enough quantities suitable for photoaffinity labeling experiments. When the ligand of interest is not intrinsically photoreactive, chemical modifications to add a photoreactive group to the parent drug, and pharmacological evaluation of these chemical modifications become necessary. With few exceptions, expression and affinity-purification of proteins are required prior to photolabeling. Methods to isolate milligram quantities of highly enriched pLGIC suitable for photoaffinity labeling experiments have been developed. In this chapter, we discuss practical aspects of experimental strategies to identify allosteric modulator binding sites in pLGIC using photoaffinity labeling.

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Authors and Affiliations

  1. Department of Neurobiology, Harvard Medical School, Boston, MA, USA

    Selwyn S. Jayakar & David C. Chiara

  2. Department of Pharmaceutical Sciences, College of Pharmacy, Texas A&M Health Sciences Center, Kingsville, TX, USA

    Gordon Ang & Ayman K. Hamouda

  3. Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M Health Science Center, Bryan, TX, USA

    Ayman K. Hamouda

  4. Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M Health Science Center, Kingsville, TX, USA

    Ayman K. Hamouda

Authors
  1. Selwyn S. Jayakar
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  2. Gordon Ang
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  3. David C. Chiara
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  4. Ayman K. Hamouda
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Corresponding author

Correspondence to Ayman K. Hamouda .

Editor information

Editors and Affiliations

  1. Department of Psychiatry, University of Florida McKnight Brain Institute, Gainesville, Florida, USA

    Firas H. Kobeissy

  2. Department of Cell Biology, Microbiology, & Molecular Biology, University of South Florida, Tampa, Florida, USA

    Stanley M. Stevens, Jr.

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Jayakar, S.S., Ang, G., Chiara, D.C., Hamouda, A.K. (2017). Photoaffinity Labeling of Pentameric Ligand-Gated Ion Channels: A Proteomic Approach to Identify Allosteric Modulator Binding Sites. In: Kobeissy, F., Stevens, Jr., S. (eds) Neuroproteomics. Methods in Molecular Biology, vol 1598. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6952-4_7

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  • DOI: https://doi.org/10.1007/978-1-4939-6952-4_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6950-0

  • Online ISBN: 978-1-4939-6952-4

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