This unit provides guidelines on how to use steady-state fluorescence spectroscopy for the quantification of protein-protein interactions. The fluorescence of a protein is characterized by its excitation and emission spectra, quantum yield, and anisotropy. These parameters can change upon interaction with another protein and can be used to measure the extent of complex formation. The source of fluorescence can be an intrinsic fluorophore, such as tryptophan or tyrosine; a covalently attached fluorescent dye; or a fluorescent binding partner, such as a nucleotide or cofactor, that interacts specifically with the complex. Protocols are provided in this unit for determining affinity constants and stoichiometry values for protein-protein interactions using equilibrium titration experiments. In addition, fluorescent labeling of proteins is discussed, and an introduction to data analysis is provided. Most of the topics addressed in this unit can easily be applied to other spectroscopic methods or to the analysis of protein-ligand interactions.
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