Measuring Multivalent Binding Interactions by Isothermal Titration Calorimetry

Tarun K. Dam, Melanie L. Talaga, Ni Fan, Curtis F. Brewer

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

Multivalent glycoconjugate-protein interactions are central to many important biological processes. Isothermal titration calorimetry (ITC) can potentially reveal the molecular and thermodynamic basis of such interactions. However, calorimetric investigation of multivalency is challenging. Binding of multivalent glycoconjugates to proteins (lectins) often leads to a stoichiometry-dependent precipitation process due to noncovalent cross-linking between the reactants. Precipitation during ITC titration severely affects the quality of the baseline as well as the signals. Hence, the resulting thermodynamic data are not dependable. We have made some modifications to address this problem and successfully studied multivalent glycoconjugate binding to lectins. We have also modified the Hill plot equation to analyze high quality ITC raw data obtained from multivalent binding. As described in this chapter, ITC-driven thermodynamic parameters and Hill plot analysis of ITC raw data can provide valuable information about the molecular mechanism of multivalent lectin-glycoconjugate interactions. The methods described herein revealed (i) the importance of functional valence of multivalent glycoconjugates, (ii) that favorable entropic effects contribute to the enhanced affinities associated with multivalent binding, (iii) that with the progression of lectin binding, the microscopic affinities of the glycan epitopes of a multivalent glycoconjugate decrease (negative cooperativity), (iv) that lectin binding to multivalent glycoconjugates, especially to mucins, involves internal diffusion jumps, (bind and jump) and (v) that scaffolds of glycoconjugates influence their entropy of binding.

Original languageEnglish (US)
Pages (from-to)71-95
Number of pages25
JournalMethods in Enzymology
Volume567
DOIs
StatePublished - 2016

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Keywords

  • Binding
  • Calorimetry
  • Enthalpy
  • Entropy
  • Glycoprotein
  • Lectin
  • Mucin
  • Multivalency
  • Scaffold
  • Stoichiometry

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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