Analysis of Protein-DNA Interactions Using Isothermal Titration Calorimetry: Successes and Failures.

Abstract

Isothermal titration calorimetry (ITC) is a golden standard for the characterization of protein-DNA binding affinities and allows direct assessment of the accompanying thermodynamic driving forces. Their interpretation can give insight into role of electrostatics, specificity of the DNA recognition, contribution of protein folding upon DNA binding and help to distinguish between minor and major groove binders. The main advantages of ITC are that the binding is measured in solution, and it requires no labeling of the samples, however, the method is not well suited for high-performance studies. Here we describe the sample preparation, a procedure to perform a typical ITC experiment, data analysis, and lastly discuss how to interpret the obtained thermodynamic parameters. In conclusion, we show examples of several unsuccessful ITC experiments and identify the underlying reasons for failed experiments. In most cases with a proper adjustment of the experimental setup, it was possible to obtain data appropriate for further analysis.