Titration Microcalorimetry

Michael L. Doyle1

1 SmithKline Beecham Pharmaceuticals, King of Prussia
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 20.4
DOI:  10.1002/0471140864.ps2004s18
Online Posting Date:  May, 2001
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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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Table of Contents

  • 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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Materials

Basic Protocol 1: Determining a Molar Ratio, Observed Affinity (Kdobs), and Observed Binding Enthalpy Change (ΔHobs) by ITC

  Materials
  • Proteins and ligands of interest
  • Appropriate pH buffer
  • Isothermal titration calorimeter (MicroCal, Calorimetry Sciences, Thermometric AB)
  • 2.5‐ml Hamilton gas‐tight syringe with blunt‐end needle long enough to reach volume of sample cell
  • UV‐visible spectrophotometer
  • Micro combination pH electrode
  • Degassing apparatus
  • Nonlinear least‐squares regression software (generally provided by the instrument manufacturer)
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Figures

Videos

Literature Cited

Literature Cited
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   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.
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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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