Thermodynamics of Molecular Recognition by Calorimetry

Abstract

When Otto von Guericke, stimulated by the previous work of Galileo and Torricelli, constructed the world’s first-ever vacuum pump in 1650 to disprove Aristotle's supposition that “nature abhors a vacuum”, he could not imagine the newly-born scientific field would get us closer to understand one of the oldest questions in the history of mankind: what is life?. Even though nobody is able to answer this question correctly yet, thermodynamics helps us to address another one, equally important: how does it work? The cellular machinery is a highly complex system, probably the most complex ever created by nature. A perfect gear with thousands of chemical reactions taking place synchronously requiring high efficiency enzymes, which are responsible for providing the cell in time with the products it needs. One of the basic aims of the biophysical research is to be able to control how enzymes work. But there's no possible control if you don't previously understand how the molecular recognition between ligand and protein occurs and how favorable it is. Thermodynamics is the only scientific field allowing to address the matter. Any molecular recognition process, as a chemical reaction, is associated with a change in the molecular properties of the reactants. Understanding the molecular recognition processes between small ligands and biological macromolecules takes a complete characterization of the binding energetic, as well as the correlation between thermodynamic data and chemical structure. Techniques such as fluorimetry, spectrophotometry or circular dichroism are convenient, fast and low sample-consuming, but their application is not universal. However, there is such a universal technique, the Isothermal Titration Calorimetry (ITC), standing above the others. Modern isothermal titration calorimeters (e.g. VP-ITC or iTC-200 from Microcal (http://www.microcal.com/) and nano ITC (http://www. tainstruments.com/) are able to measure the energetic of ligand binding (for example, a drug) in a highly reliable, fast and accurate way, using relatively small amounts of material. Typically, these calorimeters require less than 500 μg of protein per complete calorimetric titration and can measure heat effects as small as 0.1 μcal, thus allowing the determination of binding constants as large as 108 to 109 M-1. Chemical interaction changes are always associated with a heat energy exchange with the environment. This fact turns ITC, among the possible choices, in the safest bet to address these studies. In an ITC experiment the heat