Journal of Computer-Aided Molecular Design

, Volume 29, Issue 9, pp 867–883 | Cite as

Thermodynamics of protein–ligand interactions as a reference for computational analysis: how to assess accuracy, reliability and relevance of experimental data

Article

Abstract

For a conscientious interpretation of thermodynamic parameters (Gibbs free energy, enthalpy and entropy) obtained by isothermal titration calorimetry (ITC), it is necessary to first evaluate the experimental setup and conditions at which the data were measured. The data quality must be assessed and the precision and accuracy of the measured parameters must be estimated. This information provides the basis at which level discussion of the data is appropriate, and allows insight into the significance of comparisons with other data. The aim of this article is to provide the reader with basic understanding of the ITC technique and the experimental practices commonly applied, in order to foster an appreciation for how much measured thermodynamic parameters can deviate from ideal, error-free values. Particular attention is paid to the shape of the recorded isotherm (c-value), the influence of the applied buffer used for the reaction (protonation reactions, pH), the chosen experimental settings (temperature), impurities of protein and ligand, sources of systematic errors (solution concentration, solution activity, and device calibration) and to the applied analysis software. Furthermore, we comment on enthalpy–entropy compensation, heat capacities and van’t Hoff enthalpies.

Keywords

Isothermal titration calorimetry Data quality and accuracy Good measuring practice Data interpretation and correlation Heat of ionization van’t Hoff evaluation 

Supplementary material

10822_2015_9867_MOESM1_ESM.pdf (970 kb)
Experimental details are given for the ITC titrations of the analysis of the heat of ionization (Fig. 3), for the titration at different salt concentrations (Fig. 4) and for the titrations applying different protein concentrations (Fig. 5). All raw ITC thermograms and their resulting isotherms as well as the extracted parameters are listed. (PDF 969 kb)

References

  1. 1.
    Klebe G (2015) The use of thermodynamic and kinetic data in drug discovery: Decisive insight or increasing the puzzlement? ChemMedChem 10:229–231CrossRefGoogle Scholar
  2. 2.
    Kramer C, Lewis R (2012) QSARs, data and error in the modern age of drug discovery. Curr Top Med Chem 12:1896–1902CrossRefGoogle Scholar
  3. 3.
    Kramer C, Kalliokoski T, Gedeck P, Vulpetti A (2012) The experimental uncertainty of heterogeneous public K(i) data. J Med Chem 55:5165–5173CrossRefGoogle Scholar
  4. 4.
    Kalliokoski T, Kramer C, Vulpetti A (2013) Quality issues with public domain chemogenomics data. Mol Inform 32:898–905CrossRefGoogle Scholar
  5. 5.
    Wätzig H, Oltmann-Norden I, Steinicke F, Alhazmi HA, Nachbar M, El-Hady DA, Albishri HM, Baumann K, Exner T, Böckler FM, El Deeb S (2015) Data quality in drug discovery: the role of analytical performance in ligand binding assays. J Comput Aided Mol Des. doi:10.1007/s10822-015-9851-6 Google Scholar
  6. 6.
    Tanford C (1978) The hydrophobic effect and the organization of living matter. Science 200:1012–1018CrossRefGoogle Scholar
  7. 7.
    Pethica BA (2015) Misuse of thermodynamics in the interpretation of isothermal titration calorimetry data for ligand binding to proteins. Anal Biochem 472:21–29CrossRefGoogle Scholar
  8. 8.
    Wiseman T, Williston S, Brandts JF, Lin LN (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem 179:131–137CrossRefGoogle Scholar
  9. 9.
    Leavitt S, Freire E (2001) Direct measurement of protein binding energetics by isothermal titration calorimetry. Curr Opin Struct Biol 11:560–566CrossRefGoogle Scholar
  10. 10.
    Thomson JA, Ladbury JE (2004) Part II, Isothermal titration calorimetry: a tutorial. In: Ladbury JE, Doyle ML (eds) Biocalorimetry 2: application of calorimtry in the biological sciences, chap 2. Wiley, ChichesterGoogle Scholar
  11. 11.
    Perozzo R, Folkers G, Scapozza L (2004) Thermodynamics of protein–ligand interactions: history, presence, and future aspects. J Recept Signal Transduct Res 24:1–52CrossRefGoogle Scholar
  12. 12.
    Freyer MW, Lewis EA (2008) Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions. Methods Cell Biol 84:79–113CrossRefGoogle Scholar
  13. 13.
    Martin SF, Clements JH (2013) Correlating structure and energetics in protein–ligand interactions: paradigms and paradoxes. Annu Rev Biochem 82:267–293CrossRefGoogle Scholar
  14. 14.
    Klebe G (2015) Applying thermodynamic profiling in lead finding and optimization. Nat Rev Drug Discov 14:95–110CrossRefGoogle Scholar
  15. 15.
    Biela A, Sielaff F, Terwesten F, Heine A, Steinmetzer T, Klebe G (2012) Ligand binding stepwise disrupts water network in thrombin: enthalpic and entropic changes reveal classical hydrophobic effect. J Med Chem 55:6094–6110CrossRefGoogle Scholar
  16. 16.
    Freire E (2008) Do enthalpy and entropy distinguish first in class from best in class? Drug Discov Today 13:869–874CrossRefGoogle Scholar
  17. 17.
    Ladbury JE, Klebe G, Freire E (2010) Adding calorimetric data to decision making in lead discovery: a hot tip. Nat Rev Drug Discov 9:23–27CrossRefGoogle Scholar
  18. 18.
    Ferenczy GG, Keseru GM (2010) Thermodynamics guided lead discovery and optimization. Drug Discov Today 15:919–932CrossRefGoogle Scholar
  19. 19.
    Rühmann E, Betz M, Fricke M, Heine A, Schäfer M, Klebe G (2015) Thermodynamic signatures of fragment binding: validation of direct versus displacement ITC titrations. Biochim Biophys Acta 1850:647–656CrossRefGoogle Scholar
  20. 20.
    Ruehmann E, Betz M, Heine A, Klebe G (2015) Fragments can bind either more enthalpy or entropy-driven: crystal structures and residual hydration pattern suggest why. J Med Chem. doi:10.1021/acs.jmedchem.5b00812 Google Scholar
  21. 21.
    Geschwindner S, Ulander J, Johansson P (2015) Ligand binding thermodynamics in drug discovery: Still a hot tip? J Med Chem. doi:10.1021/jm501511f Google Scholar
  22. 22.
    MicroCal LLC (2004) ITC data analysis in Origin® tutorial guide. MicroCal LLC, NorthamptonGoogle Scholar
  23. 23.
    Le VH, Buscaglia R, Chaires JB, Lewis EA (2013) Modeling complex equilibria in isothermal titration calorimetry experiments: thermodynamic parameters estimation for a three-binding-site model. Anal Biochem 434:233–241CrossRefGoogle Scholar
  24. 24.
    GE Healthcare Life Sciences (2012) Microcal ITC200 system user manual 29017607 AA. GE Healthcare Bio-Sciences AB, UppsalaGoogle Scholar
  25. 25.
    Cubrilovic D, Zenobi R (2013) Influence of dimehylsulfoxide on protein–ligand binding affinities. Anal Chem 85:2724–2730CrossRefGoogle Scholar
  26. 26.
    Ghai R, Falconer RJ, Collins BM (2012) Applications of isothermal titration calorimetry in pure and applied research—survey of the literature from 2010. J Mol Recognit 25:32–52CrossRefGoogle Scholar
  27. 27.
    Biswas T, Tsodikov OV (2010) An easy-to-use tool for planning and modeling a calorimetric titration. Anal Biochem 406:91–93CrossRefGoogle Scholar
  28. 28.
    Broecker J, Vargas C, Keller S (2011) Revisiting the optimal c value for isothermal titration calorimetry. Anal Biochem 418:307–309CrossRefGoogle Scholar
  29. 29.
    Turnbull WB (2011) Divided we fall? Studying low-affinity fragments of ligands by ITC. GE Healthcare Bio-Sciences AB, UppsalaGoogle Scholar
  30. 30.
    Turnbull WB, Daranas AH (2003) On the value of c: Can low affinity systems be studied by isothermal titration calorimetry? J Am Chem Soc 125:14859–14866CrossRefGoogle Scholar
  31. 31.
    Hansen LD, Fellingham GW, Russell DJ (2011) Simultaneous determination of equilibrium constants and enthalpy changes by titration calorimetry: methods, instruments, and uncertainties. Anal Biochem 409:220–229CrossRefGoogle Scholar
  32. 32.
    Tellinghuisen J (2012) Designing isothermal titration calorimetry experiments for the study of 1:1 binding: problems with the “standard protocol”. Anal Biochem 424:211–220CrossRefGoogle Scholar
  33. 33.
    Tellinghuisen J (2005) Optimizing experimental parameters in isothermal titration calorimetry. J Phys Chem B 109:20027–20035CrossRefGoogle Scholar
  34. 34.
    Mizoue LS, Tellinghuisen J (2004) The role of backlash in the “first injection anomaly” in isothermal titration calorimetry. Anal Biochem 326:125–127CrossRefGoogle Scholar
  35. 35.
    Tellinghuisen J (2008) Isothermal titration calorimetry at very low c. Anal Biochem 373:395–397CrossRefGoogle Scholar
  36. 36.
    Tellinghuisen J (2007) Optimizing experimental parameters in isothermal titration calorimetry: variable volume procedures. J Phys Chem B 111:11531–11537CrossRefGoogle Scholar
  37. 37.
    Zhang YL, Zhang ZY (1998) Low-affinity binding determined by titration calorimetry using a high-affinity coupling ligand: a thermodynamic study of ligand binding to protein tyrosine phosphatase 1B. Anal Biochem 261:139–148CrossRefGoogle Scholar
  38. 38.
    Velazquez-Campoy A, Freire E (2006) Isothermal titration calorimetry to determine association constants for high-affinity ligands. Nat Protoc 1:186–191CrossRefGoogle Scholar
  39. 39.
    Chodera JD, Mobley DL (2013) Entropy-enthalpy compensation: role and ramifications in biomolecular ligand recognition and design. Annu Rev Biophys 42:121–142CrossRefGoogle Scholar
  40. 40.
    Cornish-Bowden A (2002) Enthalpy–entropy compensation: a phantom phenomenon. J Biosci 27:121–126CrossRefGoogle Scholar
  41. 41.
    Dunitz JD (1995) Win some, lose some: enthalpy–entropy compensation in weak intermolecular interactions. Chem Biol 2:709–712CrossRefGoogle Scholar
  42. 42.
    Olsson TSG, Ladbury JE, Pitt WR, Williams MA (2011) Extent of enthalpy–entropy compensation in protein–ligand interactions. Protein Sci 20:1607–1618CrossRefGoogle Scholar
  43. 43.
    Reynolds CH, Holloway MK (2011) Thermodynamics of ligand binding and efficiency. ACS Med Chem Lett 2:433–437CrossRefGoogle Scholar
  44. 44.
    Liu T, Lin Y, Wen X, Jorissen RN, Gilson MK (2007) BindingDB: a web-accessible database of experimentally determined protein–ligand binding affinities. Nucleic Acids Res 35:198–201CrossRefGoogle Scholar
  45. 45.
    Olsson TSG, Williams MA, Pitt WR, Ladbury JE (2008) The thermodynamics of protein–ligand interaction and solvation: insights for ligand design. J Mol Biol 384:1002–1017CrossRefGoogle Scholar
  46. 46.
    Li L, Dantzer JJ, Nowacki J, O’Callaghan BJ, Meroueh SO (2008) PDBcal: a comprehensive dataset for receptor–ligand interactions with three-dimensional structures and binding thermodynamics from isothermal titration calorimetry. Chem Biol Drug Des 71:529–532CrossRefGoogle Scholar
  47. 47.
    Myszka DG, Abdiche YN, Arisaka F, Byron O, Eisenstein E, Hensley P, Thomson JA, Lombardo CR, Schwarz F, Stafford W, Doyle ML (2003) The ABRF-MIRG’02 study: assembly state, thermodynamic, and kinetic analysis of an enzyme/inhibitor interaction. J Biomol Tech 14:247–269Google Scholar
  48. 48.
    Krimmer SG, Betz M, Heine A, Klebe G (2014) Methyl, ethyl, propyl, butyl: futile but not for water, as the correlation of structure and thermodynamic signature shows in a congeneric series of thermolysin inhibitors. ChemMedChem 9:833–846CrossRefGoogle Scholar
  49. 49.
    Jelesarov I, Bosshard HR (1999) Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to invesitigate the energetics of biomolecular recognition. J Mol Recognit 12:3–18CrossRefGoogle Scholar
  50. 50.
    Doyle ML, Louie GL, Dal Monte PR, Sokoloski TD (1995) Tight binding affinities determined from linkage to protons by titration calorimetry. Methods Enzymol 259:183–194CrossRefGoogle Scholar
  51. 51.
    Baker BM, Murphy KP (1996) Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry. Biophys J 71:2049–2055CrossRefGoogle Scholar
  52. 52.
    Parker MH, Lunney EA, Ortwine DF, Pavlovsky AG, Humblet C, Brouillette CG (1999) Analysis of the binding of hydroxamic acid and carboxylic acid inhibitors to the stromelysin-1 (matrix metalloproteinase-3) catalytic domain by isothermal titration calorimetry. Biochemistry 38:13592–13601CrossRefGoogle Scholar
  53. 53.
    Goldberg RN, Kishore N, Lennen RM (2002) Thermodynamic quantities for the ionization reaction of buffers. J Phys Chem Ref Data 31:231–370CrossRefGoogle Scholar
  54. 54.
    Keller S, Vargas C, Zhao H, Piszczek G, Brautigam CA, Schuck P (2012) High-precision isothermal titration calorimetry with automated peak shape analysis. Anal Chem 84:5066–5073CrossRefGoogle Scholar
  55. 55.
    Baum B, Muley L, Heine A, Smolinski M, Hangauer D, Klebe G (2009) Think twice: understanding the high potency of bis(phenyl)methane inhibitors of thrombin. J Mol Biol 391:552–564CrossRefGoogle Scholar
  56. 56.
    Grüner S, Neeb M, Barandun LJ, Sielaff F, Hohn C, Kojima S, Steinmetzer T, Diederich F, Klebe G (2014) Impact of protein and ligand impurities on ITC-derived protein–ligand thermodynamics. Biochim Biophys Acta 1840:2843–2850CrossRefGoogle Scholar
  57. 57.
    Boström M, Williams DRM, Ninham BW (2003) Specific ion effects: why the properties of lysozyme in salt solutions follow a Hofmeister series. Biophys J 85:686–694CrossRefGoogle Scholar
  58. 58.
    Xie D, Gulnik S, Collins L, Gustchina E, Suvorov L, Erickson JW (1997) Dissection of the pH dependence of inhibitor binding energetics for an aspartic protease: direct measurement of the protonation states of the catalytic aspartic acid residues. Biochemistry 36:16166–16172CrossRefGoogle Scholar
  59. 59.
    Chu AH, Turner BW, Ackers GK (1984) Effects of protons on the oxygenation-linked subunit assembly in human hemoglobin. Biochemistry 23:604–617CrossRefGoogle Scholar
  60. 60.
    Biela A, Nasief NN, Betz M, Heine A, Hangauer D, Klebe G (2013) Dissecting the hydrophobic effect on the molecular level: the role of water, enthalpy, and entropy in ligand binding to thermolysin. Angew Chem Int Ed 52:1822–1828CrossRefGoogle Scholar
  61. 61.
    Neeb M, Betz M, Heine A, Barandun LJ, Hohn C, Diederich F, Klebe G (2014) Beyond affinity: enthalpy–entropy factorization unravels complexity of a flat structure-activity relationship for inhibition of a tRNA-modifying enzyme. J Med Chem 57:5566–5578CrossRefGoogle Scholar
  62. 62.
    Gibb CLD, Oertling EE, Velaga S, Gibb BC (2015) Thermodynamic profiles of salt effects on a host-guest system: new insight into the hofmeister effect. J Phys Chem B. doi:10.1021/acs.jpcb.5b01708 Google Scholar
  63. 63.
    Gibb CLD, Gibb BC (2011) Anion binding to hydrophobic concavity is central to the salting-in effects of hofmeister chaotropes. J Am Chem Soc 133:7344–7347CrossRefGoogle Scholar
  64. 64.
    Fox JM, Kang K, Sherman W, Héroux A, Sastry M, Baghbanzadeh M, Lockett MR, Whitesides GM (2015) Interactions between hofmeister anions and the binding pocket of a protein. J Am Chem Soc . doi:10.1021/jacs.5b00187 Google Scholar
  65. 65.
    Spolar RS, Record MT (1994) Coupling of local folding to site-specific binding of proteins to DNA. Science 26:777–784CrossRefGoogle Scholar
  66. 66.
    Gohlke H, Klebe G (2002) Approaches to the description and prediction of the binding affinity of small-molecule ligands to macromolecular receptors. Angew Chem Int Ed 41:2644–2676CrossRefGoogle Scholar
  67. 67.
    Horn JR, Russell D, Lewis EA, Murphy KP (2001) Van’t Hoff and calorimetric enthalpies from isothermal titration calorimetry: Are there significant discrepancies? Biochemistry 40:1774–1778CrossRefGoogle Scholar
  68. 68.
    Mizoue LS, Tellinghuisen J (2004) Calorimetric vs. van’t Hoff binding enthalpies from isothermal titration calorimetry: Ba2+-crown ether complexation. Biophys Chem 110:15–24CrossRefGoogle Scholar
  69. 69.
    Pan AC, Borhani DW, Dror RO, Shaw DE (2013) Molecular determinants of drug-receptor binding kinetics. Drug Discov Today 18:667–673CrossRefGoogle Scholar
  70. 70.
    Tellinghuisen J, Chodera JD (2011) Systematic errors in isothermal titration calorimetry: concentrations and baselines. Anal Biochem 414:297–299CrossRefGoogle Scholar
  71. 71.
    Baranauskiene L, Petrikaite V, Matuliene J, Matulis D (2009) Titration calorimetry standards and the precision of isothermal titration calorimetry data. Int J Mol Sci 10:2752–2762CrossRefGoogle Scholar
  72. 72.
    Wadsö I (2000) Needs for standards in isothermal microcalorimetry. Thermochim Acta 347:73–77CrossRefGoogle Scholar
  73. 73.
    Wadsö I, Wadsö L (2005) Systematic errors in isothermal micro- and nanocalorimetry. J Therm Anal Calorim 82:553–558CrossRefGoogle Scholar
  74. 74.
    Hermans J, Barry L (2014) Equilibrium and kinetics of biological macromolecules. Wiley, HobokenGoogle Scholar
  75. 75.
    Tellinghuisen J (2004) Volume errors in isothermal titration calorimetry. Anal Biochem 333:405–406CrossRefGoogle Scholar
  76. 76.
    Good NE, Winget GD, Winter W, Connolly TN, Izawa S, Singh RMM (1966) Hydrogen ion buffers for biological research. Biochemistry 5:467–477CrossRefGoogle Scholar
  77. 77.
    Houtman JCD, Brown PH, Bowden B, Yamaguchi H, Appella E, Samelson LE, Schuck P (2007) Studying multisite binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: application to adaptor protein complexes in cell signaling. Protein Sci 16:30–42CrossRefGoogle Scholar
  78. 78.
    Scheuermann TH, Brautigam CA (2015) High-precision, automated integration of multiple isothermal titration calorimetric thermograms: new features of NITPIC. Methods 76:87–98CrossRefGoogle Scholar
  79. 79.
    Zhao H, Piszczek G, Schuck P (2015) SEDPHAT—a platform for global ITC analysis and global multi-method analysis of molecular interactions. Methods 76:137–148CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  1. 1.Department of Pharmaceutical ChemistryUniversity of MarburgMarburgGermany

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