Thermodynamic and kinetic analysis of bromodomain-histone interactions.

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Multiple factors are involved when selecting a technique and designing experiments to investigate emerging questions in biochemistry. Success depends on a conceptual understanding of a given spectroscopic approach and the ability to design a system to optimize the quality of the acquired data. In this chapter, we discuss fluorescence anisotropy and its application in characterizing the factors that drive the acetylation-dependent interactions between histone and bromodomain proteins. The steady-state and pre-steady-state binding events associated with biomolecular assemblies can be quantified with this technique, so long as the proper assays and binding models are developed. To accomplish this, the continuum of experimental considerations from instrumental setup and choice of fluorophore, to experimental procedures, and data analysis is described. The methodology is discussed in sufficient detail such that this chapter is a complete guide to setting up and performing fluorescence anisotropy measurements to study biomolecular interactions. A thermodynamic and kinetic analysis is performed to determine the factors that drive molecular recognition and binding affinity, resulting in the identification of an induced-folding mechanism. Copyright © 2009 Elsevier Inc. All rights reserved.

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Methods in enzymology