Titration Microcalorimetry
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- Abstract
- Table of Contents
- Materials
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- Literature Cited
Abstract
Isothermal titration calorimetry (ITC) is perhaps the most rigorous commercially available method for characterizing protein?ligand interactions. In this method, interactions are detected by the intrinsic heat (binding enthalpy) change of the reaction. The technique is applicable to native, unmodified proteins in solution. This is important for proteins that lose or change their functional behavior when chemically modified or attached to a surface. ITC is also useful for evaluating qualitative questions such whether a proposed binding interaction occurs at all, or for quantitatively measuring the concentration of functionally active protein. Finally, if executed with proper control experiments, ITC can be a rich source of thermodynamic information about the molecular binding mechanism.
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- Strategic Planning
- Basic Protocol 1: Determining a Molar Ratio, Observed Affinity (Kdobs), and Observed Binding Enthalpy Change (ΔHobs) by ITC
- Basic Protocol 2: Determining Biochemical Binding Thermodynamics: ΔG°′, ΔH°′ and ΔCp°′ by ITC
- Support Protocol 1: Measurement of Thermal Stability
- Support Protocol 2: Determination of Protein Assembly States
- Commentary
- Figures
- Tables
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Basic Protocol 1: Determining a Molar Ratio, Observed Affinity (Kdobs), and Observed Binding Enthalpy Change (ΔHobs) by ITC
Materials
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Figure 20.4.1 Isothermal titration calorimetry (ITC) data for titration of 5.7 mM 18‐crown‐6 ether with 5‐µL injections of 103 mM barium. (A ) Raw power (µcal/sec) versus time tracing. At each injection an exothermic “spike” is seen. The area under each spike is proportional to the heat of binding of barium to crown ether. The first injection is only a preliminary injection for experimental set up purposes and is ignored during data analysis. (B ) Amount of heat measured at each injection normalized to the number of moles of barium injected (kcal/mol) versus molar ratio of cumulative barium added per crown ether in the cell. Nonlinear least squares analysis of the data yields a molar ratio of 1.01 ± 0.01 barium ions per crown ether, an observed binding ΔH = −8.17 ± 0.01 kcal/mol barium, and an observed equilibrium dissociation constant K d = 290 ± 2 µM. View Image -
Figure 20.4.2 Resolvability of binding parameters from ITC data depends on the concentration of the cell reactant relative to the affinity of the interaction. Shown, as fraction injectant bound versus molar ratio of syringe reactant added per cell reactant, are simulated ITC curves at various “ C parameter” values, (following the analysis of Wiseman et al. ()). The C parameter is the ratio of concentration of reactant in the cell divided by the dissociation equilibrium constant, K d . When the concentration of cell reactant is too low ( C < 1), the data shape is very shallow and does not allow for determination of any of the interaction constants ( K d , molar ratio, or enthalpy change). When the cell reactant concentration is too high ( C > 1000), the binding molar ratio and observed enthalpy change are well determined, but there is insufficient information in the transition region to resolve the affinity constant, K d . Optimal C values to use for experiments are ∼10 to 100. View Image -
Figure 20.4.3 Example of ITC data where the affinity is too tight to allow resolution of K d obs . Note that only one data point is present in the transition region. The data is for an MAb‐antigen (CD4, D1D2 domains) interaction with a known K d °′ = 0.3 nM under these conditions, and the C value (see Fig. ) is 3000. (A ) Raw data shown as µcal/sec versus time. (B ) Integration of the raw data gives the amount of heat produced at each injection normalized to the number of moles D1D2 injected. Reproduced from Doyle and Hensley () with permission from Academic Press. View Image -
Figure 20.4.4 Observed binding enthalpy change from ITC depends on the buffer proton‐ionization enthalpy whenever binding is coupled to uptake or release of protons. Data are for a monoclonal antibody (MAb) protein–antigen (CD4) interaction (Doyle et al., ). The slope of the plot yields the number of protons coupled to the antibody‐antigen binding reaction, and the intercept yields the biochemical binding enthalpy change (Δ H °′ ) corrected for buffer ionization artifact heats. View Image -
Figure 20.4.5 Temperature dependence of the biochemical binding enthalpy change for a MAb protein‐antigen interaction (binding of CD4 to anti‐CD4 MAb CE9.1). The slope of the line yields a binding heat capacity change equal to −0.69 kcal/mol/°C. Different protein‐protein or protein‐ligand interactions will have different slope and intercept values, but all protein‐ligand binding enthalpy changes should exhibit a temperature dependence. Reproduced from Doyle and Hensley () with permission from Academic Press. View Image -
Figure 20.4.6 DSC data for the temperature dependence of the heat capacity of MAb CE9.1. Measurements were done at a scan rate 1°C/minute. The temperature‐dependence of the MAb heat capacity C p does not reveal any thermally induced transitions over the experimental temperature range of the present ITC studies, suggesting the MAb does not undergo temperature‐induced structure changes. Unfolding begins to occur at 60°C. Multiple transitions indicate multiple domains and are typical for MAb unfolding. Inset is thermal unfolding of D1D2 domains of CD4 as measured by circular dichroism. Measurements were done at a scan rate of 1 degree/minute. Plot shows ellipticity in millidegrees versus temperature. D1D2 is stable up to ∼50°C. The maximum temperature used in ITC studies (47°C) is indicated by the vertical dotted line. Reproduced from Doyle and Hensley () with permission from Academic Press. View Image -
Figure 20.4.7 Temperature‐dependence of the binding equilibrium dissociation constant for a MAb‐antigen (CD4) interaction. Solid curve is calculated from the integrated van't Hoff equation (Equation ) using the affinity constants measured at 45° and 47°C in combination with measured values of the binding ΔH°′ and ΔCp °′ . Squares represent affinities measured directly by titration calorimetry at higher temperatures. Circles represent affinity measured by an independent method by Doyle et al. (). Triangles represent affinity measured by surface plasmon resonance. Reproduced from Doyle and Hensley () with permission from Academic Press. View Image
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Literature Cited
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| Baker, B.M. and Murphy, K.P. 1996. Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry. Biophys. J. 71:2049‐2055. | |
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| Doyle, M.L. 1997. Characterization of binding interactions by isothermal titration calorimetry Curr. Opin. Biotechnol. 8:31‐35. | |
| Doyle, M.L. and Hensley, P. 1997. Experimental dissection of protein‐protein interactions in solution. Adv. Mol. Cell Biol. 22A:279‐ 237. | |
| Doyle, M.L. and Hensley, P. 1998. Tight ligand binding affinities determined from thermodynamic linkage to temperature by titration calorimetry. Methods Enzymol. 295:88‐99. | |
| Doyle, M.L., Gill, S.J., and Cusanovich, M.A. 1986. Ligand‐controlled dissociation of Chromatium vinosum cytochrome c′ Biochemistry 25:2509‐2516. | |
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| Guinto, E.R. and Di Cera, E. 1996. Large heat capacity change in a protein‐monovalent cation interaction. Biochemistry 35:8800‐8804. | |
| Koslov, A.G. and Lohman, T.M. 1998. Calorimetric studies of E. coli SSB protein‐single stranded DNA interactions: Effects of monovalent salts on binding enthalpy J. Mol. Biol. 278:999‐1014. | |
| Koslov, A.G., Lohman, T.M. 1999. Adenine base unstacking dominates the observed enthalpy and heat capacity changes for the Escherichia coli SSB tetramer binding to single‐stranded oligoadenylates. Biochemistry 38:7388‐7397. | |
| Liu, Y. and Sturtevant, J.M. 1995. Significant discrepancies between van't Hoff and calorimetric enthalpies. II. Protein Sci. 4:2559‐2561. | |
| Lohman, T.M., Overman, L.B., Ferrari, M.E., and Kozlov, A.G. 1996. A highly salt‐dependent enthalpy change for Escherichia coli SSB protein‐nucleic acid binding due to ion‐protein interactions. Biochemistry 35:5272‐5279. | |
| McKinnon, I.R., Fall, L., Parody‐Morreale, A., and Gill, S.J. 1984. A twin titration microcalorimeter for the study of biochemical reactions. Anal. Biochem. 139:134‐139. | |
| Murphy, K.P., Freire, E. 1992. Thermodynamics of structural stability and cooperative folding behavior in proteins. Adv. Protein Chem. 43:313‐61. | |
| Murphy, K.P., Xie, D., Garcia, K.C., Amzel, L.M., and Freire, E. 1993. Structural energetics of peptide recognition: Angiotensin II/antibody binding. Proteins Struct. Funct. Genet. 15:113‐120. | |
| Murphy, K.P., Freire, E., and Paterson, Y. 1995. Configurational effects in antibody‐antigen interactions studied by microcalorimetry. Proteins Struct. Funct. Genet. 21:83‐90. | |
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| Spolar, R.S. and Record, M.T., Jr. 1994. Coupling of local folding to site‐specific binding of proteins to DNA. Science 263:777‐784. | |
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| Wiesinger, H. and Hinz, H.J. 1986. Thermodynamic Data for Biochemistry and Biothermodynamics. pp. 211‐226. Springer‐Verlag, Berlin. | |
| Wiseman, T., Williston, S., Brandts, J.F., and Lin, L‐N. 1989. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179:131‐137. | |
| Wyman, J. and Gill, S.J. 1990. Binding and Linkage: The Functional Chemistry of Biological Macromolecules. University Science Books, Mill Valley, Calif. | |
| Key References | |
| Baker and Murphy, 1996. See above. | |
| A comprehensive treatise on the use of ITC to characterize protonation reactions that are coupled to ligand binding. | |
| Bhatnagar, R.S. and Gordon, J.I. 1995. Thermodynamic studies of myristoyl‐CoA‐protein n‐myristoyltransferase using isothermal titration calorimetry. Methods Enzymol. 250:467‐486. | |
| Reviews practical aspects of ITC, including experimental design for tight/weak binding interactions. | |
| Bundle, D.R. and Sigurskjold, B.W. 1994. Determination of accurate thermodynamics of binding by titration microcalorimetry. Methods Enzymol. 247:288‐304. | |
| Reviews practical and theoretical aspects of using ITC to characterize protein‐carbohydrate interactions. | |
| Chung et al., 1998. See above. | |
| A detailed structural‐thermodynamic study describing the role of water in achieving specificity, as well as the thermodynamic consequences. | |
| Connelly et al., 1994. See above. | |
| A detailed structural‐thermodynamic study describing the role of water in achieving specificity, as well as the thermodynamic consequences. | |
| Cooper, A. and Johnson, C.M. 1994. Isothermal titration microcalorimetry. Methods Mol. Biol. 22:125‐150. | |
| Reviews the fundamentals of conducting ITC studies. | |
| Fisher, H.F. and Singh, N. 1995. Calorimetric methods for interpreting protein‐ligand interactions. Methods Enzymol. 259:194‐221. | |
| A comprehensive review of the mechanics and practical issues involved in conducting ITC experiments. Includes discussion on data analysis and deconvolution of linked protons. | |
| Gomez and Freire, 1995. See above. | |
| A comprehensive thermodynamic study that dissects the individual contributions of amino acids to a peptide‐protein binding interaction. This paper points out the specific properties of protein‐ligand interactions (e.g., coupled protonation events and conformational entropies for amino acid side‐chains) that must be taken into account when using thermodynamic approaches to structure‐based drug design. | |
| Koslov and Lohman, 1998. See above. | |
| Reports the large effects that specific salts can have on the thermodynamics of protein‐nucleic acid interactions. Indicates that an accurate understanding of structure‐thermodynamic relationships in binding specificity will require characterization of salt effects. | |
| Lin, L.‐N., Li, J., Brandts, J.F., and Weiss, R.M. 1994. The serine receptor of bacterial chemotaxis exhibits half‐site saturation for serine binding. Biochemistry 33:6564‐6570. | |
| An example where ITC was done with membrane‐bound receptors. | |
| Lohman et al., 1996. See above. | |
| Reports the large effects that specific salts can have on the thermodynamics of protein‐nucleic acid interactions. Indicates that an accurate understanding of structure‐thermodynamic relationships in binding specificity will require characterization of salt effects. | |
| Spolar and Record, 1994. See above. | |
| Correlated binding thermodynamics to extent of conformational change observed by X‐ray and NMR structural analysis. | |
| Wiseman et al., 1989. See above. | |
| Describes ITC instrumentation and data analysis. | |
| Internet Resources | |
| http://www.biophysics.org/biophys/society/biohome.htm | |
| Biophysical Society home page. Refer to Biophysics On‐Line and Chapter on Thermodynamics links at this site. | |
| http://www.microcalorimetry.com/ | |
| Web site of MicroCal (manufacturer of high‐sensitivity microcalorimeters); includes background and literature references about calorimetry. | |
| http://www.calorimetry.com | |
| Web site of Calorimetry Sciences Corporation (manufacturer of high‐sensitivity microcalorimeters); includes background and literature references about calorimetry. | |
| http://www.calscorp.com | |
| Web site of Thermometric AB, Sweden (manufacturer of high‐sensitivity microcalorimeters); includes background and literature references about calorimetry. | |
| http://www.thermometric.com |
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