Abstract
Isothermal titration calorimetry allows the determination of thermodynamic parameters for the interaction between a protein and mono- or oligosaccharides in solution. For the study of protein–carbohydrate interactions, it is a robust way to determine the stoichiometry and affinity, as well as the enthalpic and entropic contributions to this interaction, without the use of labeled proteins or substrates. Here we describe a standard multiple-injection titration experiment for measuring the binding energetics between a carbohydrate-binding protein and an oligosaccharide.
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References
Bastos M, Velazquez-Campoy A (2021) Isothermal titration calorimetry (ITC): a standard operating procedure (SOP). Eur Biophys J 50:363–371
Abbott DW, Boraston AB (2012) Quantitative approaches to the analysis of carbohydrate-binding module function. Methods Enzymol 510:211–231
Deutscher MP (1990) Maintaining protein stability. Methods Enzymol 182:83–89
Stoll VS, Blanchard JS (1990) Buffers: principles and practice. Methods Enzymol 182:24–38
Turnbull WB, Daranas AH (2003) On the value of c: can low affinity systems be studied by isothermal titration calorimetry? J Am Chem Soc 125:14859–14866
Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, Hochstrasser DF (1999) Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 112:531–552
Cockburn DW, Suh C, Medina KP, Duvall RM, Wawrzak Z, Henrissat B, Koropatkin NM (2018) Novel carbohydrate binding modules in the surface anchored a-amylase of Eubacterium rectale provide a molecular rationale for the range of starches used by this organism in the human gut. Mol Microbiol 107:249–264
van Bueren AL, Higgins M, Wang D, Burke RD, Boraston AB (2007) Identification and structural basis of binding to host lung glycogen by streptococcal virulence factors. Nat Struct Mol Biol 14:76–84
Boraston AB, Healey M, Klassen J, Ficko-Blean E, Lammerts van Bueren A, Law V (2006) A structural and functional analysis of alpha-glucan recognition by family 25 and 26 carbohydrate-binding modules reveals a conserved mode of starch recognition. J Biol Chem 281:587–598
Cerqueira FM, Photenhauer AL, Doden HL, Brown AN, Abdel-Hamid AM, Morais S, Bayer EA, Wawrzak Z, Cann I, Ridlon JM, Hopkins JB, Koropatkin NM (2022) Sas20 is a highly flexible starch-binding protein in the Ruminococcus bromii cell-surface amylosome. J Biol Chem 298:101896
Kantonen SA, Henriksen NM, Gilson MK (2017) Evaluation and minimization of uncertainty in ITC binding measurements: heat error, concentration error, saturation, and stoichiometry. Biochim Biophys Acta Gen Subj 1861:485–498
Markova N, Hallen D (2004) The development of a continuous isothermal titration calorimetric method for equilibrium studies. Anal Biochem 331:77–88
Dumas P (2022) Isothermal titration calorimetry in the single-injection mode with imperfect mixing. Eur Biophys J 51:77–84
Boraston AB (2005) The interaction of carbohydrate-binding modules with insoluble non-crystalline cellulose is enthalpically driven. Biochem J 385:479–484
Boraston AB, Creagh AL, Alam MM, Kormos JM, Tomme P, Haynes CA, Warren RA, Kilburn DG (2001) Binding specificity and thermodynamics of a family 9 carbohydrate-binding module from Thermotoga maritima xylanase 10A. Biochemistry 40:6240–6247
Boraston AB, Bolam DN, Gilbert HJ, Davies GJ (2004) Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem J 382:769–781
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Brown, H.A., Koropatkin, N.M. (2023). Isothermal Titration Calorimetry for Quantification of Protein–Carbohydrate Interactions. In: Abbott, D.W., Zandberg, W.F. (eds) Carbohydrate-Protein Interactions. Methods in Molecular Biology, vol 2657. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3151-5_9
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DOI: https://doi.org/10.1007/978-1-0716-3151-5_9
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