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Lipid-Protein Interactions pp 351–368Cite as

Analyzing Transmembrane Protein and Hydrophobic Helix Topography by Dual Fluorescence Quenching

Part of the Methods in Molecular Biology book series (MIMB,volume 2003)

Abstract

The location of fluorescent groups relative to the lipid bilayer can be evaluated using fluorescence quenchers embedded in the membrane and/or dissolved in aqueous solution. Quenching can be used to define the membrane topography of membrane proteins and individual membrane-embedded hydrophobic helices by combining it with the placement of fluorescent groups, including Trp, at defined sequence positions. This chapter briefly discusses various quenching methods for studies of membrane protein topography, and provides detailed protocols for dual quencher analysis (DQA), a rapid, highly sensitive, and experimentally flexible approach in which the information gained from both a membrane-embedded and aqueous quencher is combined. The advantages of the DQA method include flexibility with regard to the bilayer compositions to which it can be applied, including membranes composed of lipids of varying head group and acyl chain compositions, as well as the ability to identify mixed populations of fluorophores residing at different depths within the bilayer.

Key words

  • Fluorescence spectroscopy
  • Fluorescence quenching
  • Nitroxides
  • Spin labels
  • 10-Doxyl nonadecane
  • DQA
  • Transmembrane helices

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Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Rowan University, Glassboro, NJ, USA

    Gregory A. Caputo

  2. Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA

    Erwin London

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  1. Gregory A. Caputo
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  2. Erwin London
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Correspondence to Erwin London .

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Editors and Affiliations

  1. Department of Biophysics, Institute of Biology, FB10 and Center for Interdisciplinary Nanostructure Science and Technology (CINSaT), University of Kassel, Kassel, Germany

    Prof. Dr. Jörg H. Kleinschmidt

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Caputo, G.A., London, E. (2019). Analyzing Transmembrane Protein and Hydrophobic Helix Topography by Dual Fluorescence Quenching. In: Kleinschmidt, J. (eds) Lipid-Protein Interactions. Methods in Molecular Biology, vol 2003. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9512-7_15

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  • DOI: https://doi.org/10.1007/978-1-4939-9512-7_15

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