Calorimetry Methods to Study Membrane Interactions and Perturbations Induced by Antimicrobial Host Defense Peptides

  • Mauricio Arias
  • Elmar J. Prenner
  • Hans J. VogelEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1548)


Biological membranes play an important role in determining the activity and selectivity of antimicrobial host defense peptides (AMPs). Several biophysical methods have been developed to study the interactions of AMPs with biological membranes. Isothermal titration calorimetry and differential scanning calorimetry (ITC and DSC, respectively) are powerful techniques as they provide a unique label-free approach. ITC allows for a complete thermodynamic characterization of the interactions between AMPs and membranes. DSC allows one to study the effects of peptide binding on the packing of the phospholipids in the membrane. Used in combination with mimetic models of biological membranes, such as phospholipid vesicles, the role of different phospholipid headgroups and distinct acyl chains can be characterized. In these protocols the use of ITC and DSC methods for the study of peptide–membrane interactions will be presented, highlighting the importance of membrane model systems selected to represent bacterial and mammalian cells. These studies provide valuable insights into the mechanisms involved in the membrane binding and perturbation properties of AMPs.

Key words

Antimicrobial peptides Calorimetry Isothermal titration calorimetry Differential scanning calorimetry Peptide–membrane interactions 


  1. 1.
    Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55:27–55CrossRefPubMedGoogle Scholar
  2. 2.
    Shai Y (2002) Mode of action of membrane active antimicrobial peptides. Biopolymers 66:236–248CrossRefPubMedGoogle Scholar
  3. 3.
    Nguyen LT, Haney EF, Vogel HJ (2011) The expanding scope of antimicrobial peptide structures and their modes of action. Trends Biotechnol 29:464–472CrossRefPubMedGoogle Scholar
  4. 4.
    Jenssen H, Hamill P, Hancock REW (2006) Peptide antimicrobial agents. Clin Microbiol Rev 19:491–511CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Epand RM, Vogel HJ (1999) Diversity of antimicrobial peptides and their mechanisms of action. Biochim Biophys Acta 1462:11–28CrossRefPubMedGoogle Scholar
  6. 6.
    Nicolas P (2009) Multifunctional host defense peptides: intracellular-targeting antimicrobial peptides. FEBS J 276:6483–6496CrossRefPubMedGoogle Scholar
  7. 7.
    Matsuzaki K, Sugishita K, Fujii N et al (1995) Molecular basis for membrane selectivity of an antimicrobial peptide, magainin 2. Biochemistry 34:3423–3429CrossRefPubMedGoogle Scholar
  8. 8.
    Matsuzaki K (2009) Control of cell selectivity of antimicrobial peptides. Biochim Biophys Acta 1788:1687–1692CrossRefPubMedGoogle Scholar
  9. 9.
    Lohner K, Sevcsik E, Pabst G (2008) Liposome-based biomembrane mimetic systems: implications for lipid-peptide interactions. In: Advances in planar lipid bilayers and liposomes. Elsevier, Amsterdam, pp 103–132Google Scholar
  10. 10.
    Jing WG, Prenner EJ, Vogel HJ et al (2005) Headgroup structure and fatty acid chain length of the acidic phospholipids modulate the interaction of membrane mimetic vesicles with the antimicrobial peptide protegrin-1. J Pept Sci 11:735–743CrossRefPubMedGoogle Scholar
  11. 11.
    Bozelli JC, Sasahara ET, Pinto MRS et al (2012) Effect of head group and curvature on binding of the antimicrobial peptide tritrpticin to lipid membranes. Chem Phys Lipids 165:365–373CrossRefPubMedGoogle Scholar
  12. 12.
    McElhaney RN (1982) The use of differential scanning calorimetry and differential thermal analysis in studies of model nad biological membranes. Chem Phys Lipids 30:229–259CrossRefPubMedGoogle Scholar
  13. 13.
    Lewis RNAH, Mannock DA, Mcelhaney RN (2007) Differential scanning calorimetry in the study of lipid phase. Practical considerations. Methods Mol Biol 400:171–195CrossRefPubMedGoogle Scholar
  14. 14.
    Mavromoustakos TM (2007) The use of differential scanning calorimetry to study drug – membrane interactions. Methods Mol Biol 400:587–600CrossRefPubMedGoogle Scholar
  15. 15.
    Cañadas O, Casals C (2013) Differential scanning calorimetry of protein-lipid interactions. In: Kleinschmidt JH (ed) Lipid-protein interaction: methods and protocols. Humana Press, New York, NY, pp 55–71CrossRefGoogle Scholar
  16. 16.
    Demetzos C (2008) Differential scanning calorimetry (DSC): a tool to study the thermal behavior of lipid bilayers and liposomal stability. J Liposome Res 18:159–173CrossRefPubMedGoogle Scholar
  17. 17.
    Spink CH (2008) Differential scanning calorimetry. Methods Cell Biol 84:115–141CrossRefPubMedGoogle Scholar
  18. 18.
    Lohner K, Prenner EJ (1999) Differential scanning calorimetry and X-ray diffraction studies of the specificity of the interaction of antimicrobial peptides with membrane-mimetic systems. Biochim Biophys Acta 1462:141–156CrossRefPubMedGoogle Scholar
  19. 19.
    Chiu MH, Prenner EJ (2011) Differential scanning calorimetry: an invaluable tool for a detailed thermodynamic characterization of macromolecules and their interactions. J Pharm Bioallied Sci 3:39–59CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Riske KA, Barroso RP, Vequi-suplicy CC et al (2009) Lipid bilayer pre-transition as the beginning of the melting process. Biochim Biophys Acta 1788:954–963CrossRefPubMedGoogle Scholar
  21. 21.
    Heimburg T (2000) A model for the lipid pretransition: coupling of ripple formation with the chain-melting transition. Biophys J 78:1154–1165CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Freyer MW, Lewis EA (2008) Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions. Methods Cell Biol 84:79–113CrossRefPubMedGoogle Scholar
  23. 23.
    Duff Jr MR, Grubbs J, Howell EE (2011) Isothermal titration calorimetry for measuring macromolecule-ligand affinity. J Vis Exp (55): 2796Google Scholar
  24. 24.
    Lewis EA, Murphy KP (2005) Isothermal titration calorimetry. In: Nienhaus GU (ed) Protein-ligands interactions methods and applications. Humana Press, Totowa, NJ, pp 1–15CrossRefGoogle Scholar
  25. 25.
    Bagheri M (2013) Synthesis and thermodynamic characterization of small cyclic antimicrobial arginine and tryptophan-rich peptides with selectivity for Gram-negative bacteria. In: Giuliani A, Rinaldi AC (eds) Antimicrobial peptides methods and protocols. Humana Press, New York, NY, pp 87–109Google Scholar
  26. 26.
    Seelig J (2004) Thermodynamics of lipid – peptide interactions. Biochim Biophys Acta 1666:40–50CrossRefPubMedGoogle Scholar
  27. 27.
    Wieprecht T, Seelig J (2002) Peptide-lipid interactions. Curr Top Membr 52:31–56CrossRefGoogle Scholar
  28. 28.
    Wenk MR, Seelig J (1998) Magainin 2 amide interaction with lipid membranes: calorimetric detection of peptide binding and pore formation. Biochemistry 37:3909–3916CrossRefPubMedGoogle Scholar
  29. 29.
    Henriksen JR, Andresen TL (2011) Thermodynamic profiling of peptide membrane interactions by isothermal titration calorimetry: a search for pores and micelles. Biophys J 101:100–109CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Klocek G, Schulthess T, Shai Y et al (2009) Thermodynamics of melittin binding to lipid bilayers. Aggregation and pore formation. Biochemistry 48:2586–2596CrossRefPubMedGoogle Scholar
  31. 31.
    Wieprecht T, Apostolov O, Beyermann M et al (1999) Thermodynamics of the alpha-helix-coil transition of amphipathic peptides in a membrane environment: implications for the peptide-membrane binding equilibrium. J Mol Biol 294:785–794CrossRefPubMedGoogle Scholar
  32. 32.
    Artimo P, Jonnalagedda M, Arnold K et al (2012) ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res 40:W597–W603CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Arias M, Jensen KV, Nguyen LT et al (2014) Hydroxy-tryptophan containing derivatives of tritrpticin: modification of antimicrobial activity and membrane interactions. Biochim Biophys Acta 1848:277–288CrossRefPubMedGoogle Scholar
  34. 34.
    Ames BN, Neufeld EF, Ginsberg V (1966) Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol 8:115–118CrossRefGoogle Scholar
  35. 35.
    Jing W, Hunter HN, Hagel J et al (2003) The structure of the antimicrobial peptide micelles Ac-RRWWRF-NH2 bound to micelles and its interactions with phospholipid bilayers. J Pept Res 61:219–229CrossRefPubMedGoogle Scholar
  36. 36.
    Fukada H, Takahashi K (1998) Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride. Proteins Struct Funct Genet 33:159–166CrossRefPubMedGoogle Scholar
  37. 37.
    Goldberg RN (2002) Thermodynamic quantities for the ionization reactions of buffers. J Phys Chem Ref Data 31:231CrossRefGoogle Scholar
  38. 38.
    Phillips GB, Dodge JT (1967) Composition of phospholipids and of phospholipid fatty acids in human red cells. J Lipid Res 8:667–675PubMedGoogle Scholar
  39. 39.
    Marr AG, Ingraham JL (1962) Effect of temperature on the composition of fatty acids in Escherichia coli. J Bacteriol 84:1260–1267PubMedPubMedCentralGoogle Scholar
  40. 40.
    Tellinghuisen J, Chodera JD (2011) Systematic errors in isothermal titration calorimetry: concentrations and baselines. Anal Biochem 414:297–299CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Mauricio Arias
    • 1
  • Elmar J. Prenner
    • 1
  • Hans J. Vogel
    • 1
    Email author
  1. 1.Biochemistry Research Group, Department of Biological SciencesUniversity of CalgaryCalgaryCanada

Personalised recommendations