Biophysical Chemistry 115 (2005) 115–124 http://www.elsevier.com/locate/biophyschem ITC in the post-genomic era...? Priceless Adria´n Vela´zquez Campoya,b,*, Ernesto Freirea aDepartment of Biology, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA bInstituto de Biocomputacio´n y Fı´sica de Sistemas Complejos (BIFI), Universidad de Zaragoza, Corona de Arago´n 42, E-50009 Zaragoza, Spain Received 28 June 2004; received in revised form 12 November 2004; accepted 10 December 2004 Available online 25 December 2004 Abstract The information available after decoding the genome of the human species and many others is opening the possibility of new approaches to target thousands of protein interactions critical for a continuously increasing list of genetic and infectious diseases and pathologies, and to understand complex regulatory pathways and interaction networks describing cell function and interrelation. There is a need for a reliable technique offering the capability of measuring accurately macromolecular interactions (e.g. protein/ligand, protein/protein, protein/nucleic acid) in the laboratory. Compared to other analytical techniques, isothermal titration calorimetry (ITC) exhibits some important advantages for characterizing intermolecular interactions and binding equilibria. ITC is suitable for characterizing both low affinity interactions (e.g. protein network regulation and natural ligands) and high affinity interactions (e.g. rational drug design). Considering the advanced technological level reached as well as the outstanding quality of the information accessible through this technique, ITC is expected to play a very prominent role in the next years in the areas of rational drug design and protein network regulation. D 2004 Elsevier B.V. All rights reserved. Keywords: Thermodynamics; Binding equilibrium; Isothermal titration calorimetry; Extreme binding affinity 1. Introduction tiny amount of heat associated with the non-covalent interaction between biological molecules [1–5]. Many experimental techniques can be used to study Calorimetry measures directly the heat associated with a binding processes. Some of them (e.g. dialysis, ultracen- given process, which, at constant pressure, is equal to the trifugation) rely on the direct determination of the different enthalpy change in that process, DH. Due to sensitivity and species present in the system (free macromolecule, free accuracy reasons, the calorimeters used to characterize ligand and macromolecule/ligand complex), whereas others binding processes belong to the category of titration (e.g. spectroscopy and calorimetry) measure a signal that it calorimeters with dynamic power compensation operating is proportional to the advance of the reaction (formation of at constant temperature (isothermal titration calorimeter, macromolecule/ligand complex). Due to practical reasons ITC). A detailed description of the instrument and the (amount of sample and time needed, mainly), the second technique can be found in the literature [1–9]. Briefly, the group constitutes the preferred one in biophysics and macromolecule solution is located inside the sample cell and biochemistry labs. Calorimetry entered a golden age in the the ligand solution in the injector syringe. A feedback early nineties, when, due to technological and methodo- control system supplies thermal power continuously to logical advances in the field, it was possible to measure the maintain the same temperature in both reference and sample cells. Any event taking place in the sample cell, usually accompanied by heat, will change the temperature in that cell and the feedback control system will modulate the * Corresponding author. Instituto de Biocomputacio´n y Fı´sica de Sistemas Complejos (BIFI), Universidad de Zaragoza, Corona de Arago´n power supplied in order to minimize such temperature 42, E-50009 Zaragoza, Spain. Tel.: +34 976 562215; fax: +34 976 562215. imbalance. A sequence of injections is programmed and the E-mail address: [email protected] (A. Vela´zquez Campoy). ligand solution is injected periodically into the sample cell. 0301-4622/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bpc.2004.12.015 116 A. Vela´zquez Campoy, E. Freire / Biophysical Chemistry 115 (2005) 115–124 The value of the association constant (or binding affinity), preparing samples, conducting experiments, analyzing data Ka, governs the equilibrium, i.e. the partition between the and discussing results, since any additional or spurious different species (free and bound). The change in compo- heat-generating process will contribute to the experimental sition inside the sample cell after each injection triggers the output. And second, enthalpy is directly measured without binding reaction and the rearrangement of populations any additional assumption. When using spectroscopic leading to the formation of complex. The system will pass techniques, experiments at different temperatures have to through different equilibrium states, differing in composi- be performed in order to estimate the binding enthalpy tion, as the sequence of injections proceeds. The heat from the temperature dependence of the association associated with each injection is proportional to the increase constant, the van’t Hoff relationship: in complex concentration (advance of the reaction) and it is BlnK DH calculated integrating the area under the deflection of the a ¼À B 1 signal measured (amount of heat per unit of time provided to T R maintain both cells, sample and reference, at the same ÀÁ temperature). At the end of the experiment, saturation of the B2lnK T 2DC a ¼ P ð3Þ macromolecule is reached. Applying non-linear regression B 1 2 R using the appropriate model in the data analysis procedure, T it is possible to estimate the association constant or binding À Á affinity, Ka, the binding enthalpy, DH, and the stoichiom- This approach exhibits two main drawbacks. First, due etry, n, in a single experiment. Spectroscopic techniques in to practical reasons, it is not always possible to cover a which the signal is also proportional to the advance of the broad temperature range when estimating the association reaction (e.g. absorbance, fluorescence intensity, molar constant. Second, although enthalpy and entropy are ellipticity or chemical shift) are only able to determine the strongly dependent on temperature, the Gibbs energy association constant in a single experiment, requiring usually exhibits a weak temperature dependency over the additional experiments and assumptions to estimate the experimentally accessible temperature range (see Fig. 1). enthalpy of binding. This enthalpy/entropy compensation phenomenon makes Calorimetry is unique in that it is capable of measuring very difficult to obtain accurately first and second simultaneously the association constant and the enthalpy of derivatives of the association constant (related to the binding. Therefore, it is possible a complete thermody- enthalpy and heat capacity of binding), both readily namic characterization of the binding process, that is, the available via calorimetry. And third, the determination of evaluation of the main thermodynamic potentials associ- the binding affinity and the Gibbs energy of binding are ated with the binding process, using basic thermodynamic accompanied of certain error. These difficulties have led to relationships: discrepancies between the enthalpy measured calorimetri- D cally (calorimetric enthalpy) and spectroscopically (van’t G ¼ÀRTlnKa Hoff enthalpy) that can be explained by errors and inaccuracies inherent to the non-calorimetric methods DG ¼ DH À TDS ð1Þ [10–14]. Let us take the temperature derivative of the where DG is the Gibbs energy change, DS is the entropy two contributions to the Gibbs energy, the enthalpy and the change, R is the gas constant and T is the absolute entropy: temperature. Enthalpy and entropy are temperature-depend- BD ent thermodynamic potentials: H D B ¼ CP T ! BDH BDS P DC ¼ ¼ T ð2Þ P BT BT P P B D T S D D D ¼ CP þ S ð4Þ where CP is the heat capacity change upon binding. BT !P 2. ITC: estimation of the enthalpy of binding If we consider now that binding entropy changes larger than F30 cal/Kd mol are unusual and heat capacity changes Heat is the direct observable in calorimetry. This fact is values between À200 and À500 cal/Kd mol are very the root for calorimetry’s strong and weak points. First, common (i.e. normally the entropy change is less than being heat a universal signal, reporter labels are not 10% of the heat capacity change and DS is negligible required to follow the reaction advance (making possible compared to DCP), we can conclude the two contributions to use natural non-modified ligands). However, only to the Gibbs energy share approximately the same temper- overall non-mechanistic information about the binding ature factor, DCP, thus canceling out the temperature process is available. Extreme care must be taken in dependency in DG. On the other hand, the temperature A. Vela´zquez Campoy, E. Freire / Biophysical Chemistry 115 (2005) 115–124 117 9 30 1.4x10 1.2x10 9 20 1.0x10 9 10 ) -1 8 S (kcal/mol) 8.0x10 (M ∆ 0 a 8 K H, T 6.0x10 ∆ -10 8 G, 4.0x10 ∆ -20 2.0x10 8 10 15 20 25 30 35 40 10 15 20 25 30 35 40 Temperature (ºC) Temperature (ºC) Fig. 1. Temperature profile of the Gibbs energy DG (solid line), the enthalpy DH (dashed line), the entropy TDS (dotted line) and the affinity Ka (dashed-dotted 9 À1 9 line) for a macromolecule/ligand binding characterized by: (left)