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Lipid-Protein Interactions pp 71–89Cite as

Thermodynamic Analysis of Protein–Lipid Interactions by Isothermal Titration Calorimetry

Part of the Methods in Molecular Biology book series (MIMB,volume 2003)

Abstract

Isothermal titration calorimetry is a highly sensitive and powerful technique for the study of molecular interactions. This method can be applied universally for studying the interaction between moleculeAbstracts, molecular assembles and ions as it measures the heat changes resulting from such interactions and does not need any probe molecule/moiety to be incorporated into the system under investigation. This method has been applied quite extensively to investigate the interaction of proteins with other biomolecules such as small ligands, other proteins, nucleic acids, lipid membranes as well as to study the interaction of antibodies, drugs, metal ions and nanoparticles with target proteins or antigens, nucleic acids, and membranes. In this chapter, we describe the application of ITC for the investigation of thermodynamics of protein–lipid interaction. A number of important parameters such as enthalpy of binding (ΔH), entropy of binding (ΔS), association constant (Ka), binding stoichiometry (n) and free energy of binding (ΔG) can be obtained from a single calorimetric titration, providing a complete thermodynamic characterization of the interaction. The method is described in detail taking the major protein of the bovine seminal plasma, PDC-109, which exhibits a high preference for interaction with choline-containing lipids, as an example. The method can be applied to investigate thermodynamic parameters associated with the interaction of other soluble proteins with lipid membranes.

Key words

  • Isothermal titration calorimetry
  • Protein–lipid interaction
  • Binding stoichiometry
  • Association constant
  • Enthalpy of binding
  • Entropy of binding

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References

  1. Ladbury JE, Chowdhry BZ (1996) Sensing the heat: the application of isothermal titration calorimetry to thermodynamic studies of biomolecular interaction. Chem Biol 3:791–801

    CAS  PubMed  Google Scholar 

  2. Pierce MM, Raman CS, Nal BT (1999) Isothermal titration calorimetry of protein-protein interactions. Methods 19:213–221

    CAS  PubMed  Google Scholar 

  3. Velázquez-Campoy A, Ohtaka H, Nezami A, Muzammil S, Freire E (2004) Isothermal titration calorimetry. Curr Protoc Cell Biol Chapter 17:Unit 17.8. https://doi.org/10.1002/0471143030.cb1708s23

    CrossRef  PubMed  Google Scholar 

  4. Weber PC, Salemme FR (2003) Applications of calorimetric methods to drug discovery and the study of protein interactions. Curr Opin Struct Biol 13:115–121

    CAS  PubMed  Google Scholar 

  5. Privalov PL, Dragan AI (2007) Microcalorimetry of biological macromolecules. Biophys Chem 126:16–24

    CAS  PubMed  Google Scholar 

  6. Buurma NJ, Haq I (2007) Advances in the analysis of isothermal titration calorimetry data for ligand–DNA interactions. Methods 42:162–172

    CAS  PubMed  Google Scholar 

  7. Freyer MW, Lewis EA (2008) Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions. Methods Cell Biol 84:79–113

    CAS  PubMed  Google Scholar 

  8. Ladbury JE (2010) Calorimetry as a tool for understanding biomolecular interactions and an aid to drug design. Biochem Soc Trans 38:888–893

    CAS  PubMed  Google Scholar 

  9. Yanaka S, Moriwaki Y, Tsumoto K, Sugase K (2017) Elucidation of potential sites for antibody engineering by fluctuation editing. Sci Rep 7:9597

    PubMed  PubMed Central  Google Scholar 

  10. de Taeye SW, Ozorowski G, de la Pena AT, Guttman M, Julien JP, van den Kerkhof TL, Burger JA, Pritchard LK, Pugach P, Yasmeen A, Crampton J, Hu J, Bontjer I, Torres JL, Arendt H, DeStefano J, Koff WC, Schuitemaker H, Eggink D, Berkhout B, Dean H, LaBranche C, Crotty S, Crispin M, Montefiori DC, Klasse PJ, Lee KK, Moore JP, Wilson IA, Ward AB, Sanders RW (2015) Immunogenicity of stabilized HIV-1 envelope trimers with reduced exposure of non-neutralizing epitopes. Cell 163:1702–1715

    PubMed  PubMed Central  Google Scholar 

  11. Sankhala RS, Lokareddy RK, Cingolani G (2016) Divergent evolution of nuclear localization signal sequences in herpes virus terminase subunits. J Biol Chem 291:11420–11433

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Sankhala RS, Lokareddy RK, Begum S, Pumroy RA, Gillilan RE, Cingolani G (2017) Three-dimensional context rather than NLS amino acid sequence determines importin subtype specificity for RCC1. Nat Commun 8:979

    PubMed  PubMed Central  Google Scholar 

  13. Wu W, Sankhala RS, Florio TJ, Zhou L, Nguyen NLT, Lokareddy RK, Cingolani G, Panté N (2017) Synergy of two low-affinity NLSs determines the high avidity of influenza A virus nucleoprotein NP for human importin alpha isoforms. Sci Rep 7:11381

    PubMed  PubMed Central  Google Scholar 

  14. Menchicchi B, Fuenzalida JP, Bobbili KB, Hensel A, Swamy MJ, Goycoolea FM (2014) The structure of chitosan determines its interactions with mucin. Biomacromolecules 15:3550–3558

    CAS  PubMed  Google Scholar 

  15. Loh W, Brinatti C, Tam KC (2016) Use of isothermal titration calorimetry to study surfactant aggregation in colloidal systems. Biochim Biophys Acta 1860:999–1016

    CAS  PubMed  Google Scholar 

  16. Gopal B, Swaminathan CP, Bhattacharya S, Bhattacharya A, Murthy MR, Surolia A (1997) Thermodynamics of metal ion binding and denaturation of a calcium binding protein from Entamoeba histolytica. Biochemistry 36:10910–10916

    CAS  PubMed  Google Scholar 

  17. De M, You CC, Srivastava S, Rotello VM (2007) Biomimetic interactions of proteins with functionalized nanoparticles: a thermodynamic study. J Am Chem Soc 129:10747–10753

    CAS  PubMed  Google Scholar 

  18. Zhang L, Zheng Y, Xi Z, Luo Z, Xu X, Wang C, Liu Y (2009) Metal ions binding to recA inteins from Mycobacterium tuberculosis. Mol Biosyst 5:644–650

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Chakraborty S, Joshi P, Shanker V, Ansari ZA, Singh SP, Chakrabarti P (2011) Contrasting effect of gold nanoparticles and nanorods with different surface modifications on the structure and activity of bovine serum albumin. Langmuir 27:7722–7731

    CAS  PubMed  Google Scholar 

  20. Huang B, Lau BLT (2016) Biomolecule-nanoparticle interaction: elucidation of the thermodynamics by isothermal titration calorimetry. Biochim Biophys Acta 1860:945–956

    CAS  PubMed  Google Scholar 

  21. Johnson RA, Manley OM, Spuches AM, Grossehme WE (2016) Dissecting ITC data of metal ions binding to ligands and proteins. Biochim Biophys Acta 1860:892–901

    CAS  PubMed  Google Scholar 

  22. Kutcherlapati SNR, Koyilapu R, Boddu UMR, Datta D, Perali RS, Swamy MJ, Jana T (2017) Glycopolymer grafted nanoparticles: synthesis using RAFT polymerization and binding study with lectin. Macromolecules 50:7309–7320

    CAS  Google Scholar 

  23. Liu S, Lockless SW (2018) Ion binding to transport proteins using isothermal titration calorimetry. Methods Mol Biol 1684:289–303

    CAS  PubMed  Google Scholar 

  24. Boudker O, Oh S (2015) Isothermal titration calorimetry of ion-coupled membrane transporters. Methods 76:171–182

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Draczkowski P, Matosiuk D, Jozwiak K (2014) Isothermal titration calorimetry in membrane protein research. J Pharm Biomed Anal 87:313–325

    CAS  PubMed  Google Scholar 

  26. Rajarathnam K, Rösgen J (2014) Isothermal titration calorimetry of membrane proteins – progress and challenges. Biochim Biophys Acta 1838:69–77

    CAS  PubMed  Google Scholar 

  27. Wöhri AB, Hillertz P, Eriksson PO, Meuller J, Dekker N, Snijder A (2013) Thermodynamic studies of ligand binding to the human homopentameric glycine receptor using isothermal titration calorimetry. Mol Membr Biol 30:169–183

    PubMed  Google Scholar 

  28. Heerklotz H (2004) The microcalorimetry of lipid membranes. J Phys Condens Matter 16:R441–R467

    CAS  Google Scholar 

  29. Cooper MA (2004) Advances in membrane receptor screening and analysis. J Mol Recognit 17:286–315

    CAS  PubMed  Google Scholar 

  30. Boggs JM, Stamp D, Moscarello MA (1981) Interaction of myelin basic protein with dipalmitoylphosphatidylglycerol: dependence on the lipid phase and investigation of a metastable state. Biochemistry 20:6066–6072

    CAS  PubMed  Google Scholar 

  31. Boggs JM, Wood DD, Moscarello MA (1981) Hydrophobic and electrostatic interactions of myelin basic proteins with lipid. Participation of N-terminal and C-terminal portions. Biochemistry 20:1065–1073

    CAS  PubMed  Google Scholar 

  32. Sankaram MB, Brophy PJ, Marsh D (1989) Selectivity of interaction of phospholipids with bovine spinal cord myelin basic protein studied by spin-label electron spin resonance. Biochemistry 28:9699–9707

    CAS  PubMed  Google Scholar 

  33. Maggio B, Sturtevant JM, Yu RK (1987) Effect of myelin basic protein on the thermotropic behavior of aqueous dispersions of neutral and anionic glycosphingolipids and their mixtures with dipalmitoylphosphatidylcholine. J Biol Chem 262:2652–2659

    CAS  PubMed  Google Scholar 

  34. Desnoyers L, Manjunath P (1992) Major proteins of bovine seminal plasma exhibit novel interactions with phospholipids. J Biol Chem 267:10149–10155

    CAS  PubMed  Google Scholar 

  35. Ramakrishnan M, Anbazhagan V, Pratap TV, Marsh D, Swamy MJ (2001) Membrane insertion and lipid-protein interactions of bovine seminal plasma protein, PDC-109 investigated by spin label electron spin resonance spectroscopy. Biophys J 81:2215–2225

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Anbazhagan V, Sankhala RS, Singh BP, Swamy MJ (2011) Isothermal titration calorimetric studies on the interaction of the major bovine seminal plasma protein, PDC-109 with phospholipid membranes. PLoS One 6:e25993

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Greube A, Müller K, Töpfer-Petersen E, Herrmann A, Müller P (2004) Interaction of Fn type II proteins with membranes: stallion seminal plasma protein SP-1/2. Biochemistry 43:464–472

    CAS  PubMed  Google Scholar 

  38. Kumar CS, Swamy MJ (2016) A pH switch regulates the inverse relationship between membranolytic and chaperone-like activities of HSP-1/2, a major protein of horse seminal plasma. Biochemistry 55:3650–3657

    CAS  PubMed  Google Scholar 

  39. Surolia A, Bachhawat BK, Podder SK (1975) Interaction between lectin from Ricinus communis and liposomes containing gangliosides. Nature 257:802–804

    CAS  PubMed  Google Scholar 

  40. Grant CW, Peters MW (1984) Lectin-membrane interactions. Information from model systems. Biochim Biophys Acta 779:403–422

    CAS  PubMed  Google Scholar 

  41. Ramalingam TS, Das PK, Podder SK (1994) Ricin-membrane interaction: membrane penetration depth by fluorescence quenching and resonance energy transfer. Biochemistry 33:12247–12254

    CAS  PubMed  Google Scholar 

  42. Diab C, Tribet C, Gohon Y, Popot JL, Winnik FM (2007) Complexation of integral membrane proteins by phosphorylcholine-based amphipols. Biochim Biophys Acta 1768:2737–2747

    CAS  PubMed  Google Scholar 

  43. Tribet C, Diab C, Dahmane T, Zoonens M, Popot JL, Winnik FM (2009) Thermodynamic characterization of the exchange of detergents and amphipols at the surfaces of integral membrane proteins. Langmuir 25:12623–12634

    CAS  PubMed  Google Scholar 

  44. Seelig J (2004) Thermodynamics of lipid-peptide interactions. Biochim Biophys Acta 1666:40–50

    CAS  PubMed  Google Scholar 

  45. MacDonald RC, MacDonald RI, Menco BPM, Takeshita K, Subbarao NK, Hu L (1991) Small-volume extrusion apparatus for preparation of large, unilamellar vesicles. Biochim Biophys Acta 1061:297–303

    CAS  PubMed  Google Scholar 

  46. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  47. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    CAS  PubMed  Google Scholar 

  48. Noble JE, Knight AE, Reason AJ, Di Matola A, Bailey MJ (2007) A comparison of protein quantitation assays for biopharmaceutical applications. Mol Biotechnol 37:99–111

    CAS  PubMed  Google Scholar 

  49. Edelhoch H (1967) Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6:1948–1954

    CAS  PubMed  Google Scholar 

  50. Gill SC, Von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326

    CAS  PubMed  Google Scholar 

  51. ITC Data Analysis in Origin®. (1998) Tutorial Guide Version 5.0

    Google Scholar 

  52. Dimitrova MN, Matsumura H, Terezova N, Neytchev V (2002) Binding of globular proteins to lipid membranes studied by isothermal titration calorimetry and fluorescence. Colloids Surf B Biointerfaces 24:53–61

    CAS  Google Scholar 

  53. Arnulphi C, Jin L, Tricerri MA, Jonas A (2004) Enthalpy-driven apolipoprotein A-I and lipid bilayer interaction indicating protein penetration upon lipid binding. Biochemistry 43:12258–12264

    CAS  PubMed  Google Scholar 

  54. 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 179:131–137

    CAS  PubMed  Google Scholar 

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Acknowledgments

M.J.S. acknowledges the Department of Science and Technology (India) for a research grant which supported the work done in his laboratory and referred to here. R.S.S. and B.P.S. thank CSIR (India) and UGC (India) for Senior Research Fellowships. We thank Dr. V. Anbazhagan for a fruitful collaboration in the ITC studies on protein–lipid interaction.

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Authors and Affiliations

  1. School of Chemistry, University of Hyderabad, Hyderabad, India

    Musti J. Swamy, Rajeshwer S. Sankhala & Bhanu Pratap Singh

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  1. Musti J. Swamy
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  2. Rajeshwer S. Sankhala
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  3. Bhanu Pratap Singh
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Editors and Affiliations

  1. Department of Biophysics, Institute of Biology, FB10 and Center for Interdisciplinary Nanostructure Science and Technology (CINSaT), University of Kassel, Kassel, Germany

    Prof. Dr. Jörg H. Kleinschmidt

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Swamy, M.J., Sankhala, R.S., Singh, B.P. (2019). Thermodynamic Analysis of Protein–Lipid Interactions by Isothermal Titration Calorimetry. In: Kleinschmidt, J. (eds) Lipid-Protein Interactions. Methods in Molecular Biology, vol 2003. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9512-7_4

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  • DOI: https://doi.org/10.1007/978-1-4939-9512-7_4

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