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Chapter 5

Fluorescence Techniques to Characterise Ligand Binding to Proteins

Fluorescence-based techniques are widely used to investigate protein/ligand interactions, mainly due to their unsurpassed sensitivity, which allows investigating interactions down to the single molecule level. Moreover, the multiplicity of fluorescence parameters enables to investigate nearly any protein/ligand interaction. Though the intrinsic fluorescence of proteins appears as a convenient tool, the limited spectroscopic properties of Trp residues frequently require the use of external fluorophores, provided that they do not affect the folding and the functions of the labeled partner, as well as the interaction process. Among the usual questions addressed to fully characterize a protein/ligand interaction, fluorescence techniques are particularly suited for identifying the ligands using high-throughput screening and characterizing the parameters that govern the binding process. The present chapter is an overview on the use of fluorescence techniques to quantitatively determine these parameters. After a description of the basic principles of fluorescence spectroscopy and, the main techniques and fluorophores used for monitoring protein/ligand interactions, we delineate the major advantages and pitfalls of fluorescence-based techniques. Next, we describe several formalisms used to determine the binding stoichiometry and constants, stressing on the limits when systems with multiple binding sites are investigated. Finally, we illustrate the use of these techniques in characterizing the binding of zinc ions and oligonucleotides to the HIV-1 nucleocapsid protein. This example shows that a binding process could be consistently investigated through different approaches and that not only the binding parameters, but also the kinetic binding parameters, the electrostatic and nonelectrostatic contributions to the binding energy, as well as the entropy and enthalpy changes could be determined.

Print publication date: 01 Apr 2011
Copyright year: 2011
Print ISBN: 978-1-84973-009-9
PDF eISBN: 978-1-84973-266-6
From the book series:
RSC Biomolecular Sciences