Received: 2 June 2008, Revised: 30 June 2008, Accepted: 30 June 2008, Published online in Wiley InterScience: 2008A sur vey of the year 2007 literature onapplications of isothermal titrationcalorimetrySasˇa Bjelic´aand Ilian Jelesarova*Elucidation of the energetic principles of binding affinity and specificity is a central task in many branches of currentsciences: biology, medicine, pharmacology, chemistry, material sciences, etc. In biomedical research, integralapproaches combining structural information with in-solution biophysical data have proved to be a powerful waytoward understanding the physical basis of vital cellular phenomena. Isothermal titration calorimetry (ITC) is avaluable experimental tool facilitating quantification of the thermodynamic parameters that characterize recognitionprocesses involving biomacromolecules. The method provides access to all relevant thermodynamic information byperforming a few experiments. In particular, ITC experiments allow to by-pass tedious and (rarely precise) proceduresaimed at determining the changes in enthalpy and entropy upon binding by van’t Hoff analysis. Notwithstandinglimitations, ITC has now the reputation of being the ‘‘gold standard’’ and ITC data are widely used to validatetheoretical predictions of thermodynamic parameters, as well as to benchmark the results of novel binding assays. Inthis paper, we discuss several publications from 2007 reporting ITC results. The focus is on applications in biologicallyoriented fields. We do not intend a comprehensive coverage of all newly accumulated information. Rather, weemphasize work which has captured our attention with originality and far-reaching analysis, or else has providedideas for expanding the potential of the method. Copyright ß 2008 John Wiley & Sons, Ltd.Keywords: thermodynamics; calorimetry; molecular recognition; ligand binding; enthalpy; entropy; heat capacityINTRODUCTIONPresent-day large-scale genomics, proteomics, interactomics andother initiatives, and efforts to establish system-orientedapproaches are expected to provide a global understanding ofbiological processes. However, many aspects of the intimatemolecular mechanisms involved in biological function remainobscure. Macromolecular recognition is a typical example.Notwithstanding the serious progress that has been achievedover the past three decades, many details about the mechanistic,energetic, and kinetic principles of binding affinity and specificityremain vaguely understood. Part of the problem is that, at least atthe structural level, there are no obvious unifying principles in thearchitecture of protein–protein and other protein/ligand com-plexes. Binding interfaces span hundreds and thousands ofsquare angstroms, yet point mutations can severely impairbinding affinity. Chemically unrelated ligands can effectivelycompete for the same binding site. It is still very difficult toachieve high affinity and specificity of a designed molecule for atarget pocket by rational design and optimization. This is whymethodologically rigorous biophysical studies of diverse protein/ligand complexes are an indispensable endeavor toward betterunderstanding of biological function. The ultimate goal is to findlinks between molecular structure, energetics, and dynamics, andto discover ‘‘rules’’ guiding the prediction of the energeticresponse of a particular complex to structural changes in thebinding partners. In research programs combining biophysicaland structural approaches, isothermal titration calorimetry (ITC)has evolved as a valuable tool.The theoretical background, experimental design, and prac-tical aspects of the ITC experiment are discussed in detail inReferences 1–7. Here, only a brief description of the technique isgiven outlining the essential features of the method. We considerthe simplest case of a 1:1 binding reaction. The ITC experimentconsists of additions of molecule L (ligand), which is placed in theinjection syringe, to molecule R (receptor), which is contained inthe reaction cell. The injection syringe rotates, thus facilitatingrapid mixing of the reactants. A reference cell that is identical inshape and volume to the reaction cell is filled with water. Bothcells are placed in an insulated jacket and are equilibrated priorthe experiment at the desired temperature. The powercompensation principle is implemented in most of the titrationcalorimeters used in biologically oriented studies nowadays.Constant power is applied to the reference cell as to maintain aminute temperature difference between the cells (DT). Uponbinding of L to R, heat is released (exothermic reaction) or(www.interscience.wiley.com) DOI:10.1002/jmr.909Review Article* Correspondence to: I. Jelesarov, Biochemisches Institut der Universita¨t Zu¨rich,Winterthurerstrasse 190, CH-8057 Zu¨rich, Switzerland.E-mail: [email protected] S. Bjelic´, I. JelesarovBiochemisches Institut der Universita¨t Zu¨rich, Winterthurerstrasse 190,CH-8057 Zu¨rich, SwitzerlandJ. Mol. Recognit. 2008; 21: 289–311 Copyright ß 2008 John Wiley & Sons, Ltd.289absorbed (endothermic reaction). Thermopile/thermocouplecircuits detect the resulting change of DT. The feedback circuitdecreases or increases the power supplied to the reaction cell inorder to keep DT constant throughout the experiment. Since thepower changes (differential power; units of J s1) are monitoredcontinuously, a peak-shaped deflection from the thermal baselineis observed. Integration of the differential power peak over timeyields the heat, q (units of J), released or absorbed upon bindingof j mol L to R. If j is known, the ratio q/j corresponds at constanttemperature and pressure to the molar enthalpy of binding,DH (J mol1). In practice, however, the number of bound molesL(j––[L]bound) is unknown if the binding constant, KA, is unknown.Therefore, a titration experiment is required to determine KAandDH. A series of additions of L to R is per formed, so that the ratio ofthe total concentrations [L]tot/[R]totincreases from <0.1 to >2–3(or more). The observed heats monitor the extent of binding asthe degree of saturation increases. After corrections for theunspecific heats of dilution, for the changes in concentrations ofL and R, and the displacement of part of the reac tants from theactive volume of the cell, the heat detected in each injection isproportional to the molar enthalpy according to:q ¼ VcellDHR½totYi Yi1ðÞ (1)Vcellis the cell