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Fluorescence Techniques in Analysis of Protein–Ligand Interactions

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Protein-Ligand Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1008))

Abstract

Fluorescence spectroscopy may serve as a universal tool for the study of protein–ligand interactions. Applications of fluorometry have made use of various aspects of fluorescence such as intensity, emission and excitation spectra, lifetime, quantum yield, polarization state, and anisotropy, as well as energy transfer and other electronic phenomena. An experimentalist has to consider each of these characteristics carefully, frequently in combination with each other, for the analysis of protein–ligand complexes and for the determination of binding constants. Most of the available techniques are of a rather general nature and a wealth of possibilities exists for their utilization. In this chapter we will provide a short survey of selected techniques that can be used for measuring binding constants and probing protein–ligand interactions. Basic principles and phenomena are discussed followed by experimental considerations and examples of binding constant determination. Emphasis is placed on steady-state techniques that employ the use of intrinsic protein fluorescence, labeled ligands, as well as anisotropy and resonance energy transfer.

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Acknowledgments

This publication was made possible in part by grant no. G12RR003061 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the author and do not necessarily represent the official view of NCRR or NIH (GM). This work was supported by National Institutes of Health grants RO1GM076665 (JAR). We thank Professor David M. Jameson for useful discussions and for proofreading the manuscript.

Author information

Authors and Affiliations

  1. Pacific Biosciences Research Center, University of Hawaii, Honolulu, HI, USA

    Gabor Mocz

  2. Queensland Institute of Medical Research, Herston, QLD, Australia

    Justin A. Ross

Authors
  1. Gabor Mocz
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  2. Justin A. Ross
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Editor information

Editors and Affiliations

  1. Birbeck, University of London, ISMB Biophysics Centre, Institute of Str, Malet Street, London, WC1E 7HX, United Kingdom

    Mark A. Williams

  2. Birkbeck, University of London, School of Crystallography, ISMB Biophysics Centre, Institute of Str, Malet Street, London, WC1E 7HX, United Kingdom

    Tina Daviter

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Mocz, G., Ross, J.A. (2013). Fluorescence Techniques in Analysis of Protein–Ligand Interactions. In: Williams, M., Daviter, T. (eds) Protein-Ligand Interactions. Methods in Molecular Biology, vol 1008. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-398-5_7

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  • DOI: https://doi.org/10.1007/978-1-62703-398-5_7

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-397-8

  • Online ISBN: 978-1-62703-398-5

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