Isothermal titration proteins

T. K. Dam and C. F. Brewer, Multivalent protein-carbohydrate interactions Isothermal titration microcalorimetry studies, Methods Enzymol., 379 (2004) 107-128. 

Baker, B.M. and K.P. Murphy. 1996. Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry. Biophys J 71 2049-2055. 

Frazier, R.A., Papadopoulou, A., Mueller-Harvey, I., Kissoon, D., and Green, R.J., Probing protein-tannin interactions by isothermal titration microcalorimetry, J. Agric. Food Chem., 51, 5189, 2003. 

The energetics of protein association can be studied by a variety of experimental techniques,17 each of which permits measurements of equilibrium or kinetic values in a certain range. Widely used techniques include isothermal titration calorimetry, surface plasmon resonance measurement, stopped flow kinetics, optical spectroscopy, MS, and analytical ultracentrifugation. The techniques differ in their requirements (e.g., amount of protein, labeling with fluorophores, attachment to sensor surfaces, and the environment provided by the experimental set up) and therefore in their applicability to individual cases. Different techniques can also give quite different values for what might be expected to be the same quantity. For example, association rates measured by surface plasmon resonance, with one protein immobilized on a surface, are usually different from those measured for the two proteins in solution and under otherwise similar conditions. 

Protonation Effects in Protein Binding Reactions Using Isothermal Titration Calorimetry. 

The traditional methods of evaluating the interactions of glycans and glycan-binding proteins are based on isothermal titration calorimetry, surface plasmon resonance (SPR), and enzyme-linked lectin assays. Recently, glycan arrays have been developed as a... 

Pierce, M. M., Raman, C. S., Nall, B. T. (1999). Isothermal titration calorimetry of protein-protein interactions. Methods 19, 213-221. 

If a three-dimensional structure of the PTP is available, in silico docking can be utilized to model and dock the inhibitor into the active site (Fig. 5). The docking can then be verified by mutagenesis studies, in which specific amino acids that the docking predicted to interact with the inhibitor are mutated to alanine. Inhibitory activity of the compound is then measured with the mutant protein and is expected to be lower than with the wild-type protein (Fig. 4c). Finally, biophysical binding studies, such as isothermal titration calorimetry (ITC) [33] or ThermoFluor [34] may further aid in prioritizing compounds for cell-based assays and high-resolution structural analysis. 

Whereas the binding of CBMs to insoluble carbohydrates can be monitored by the UV absorption of the protein, relatively large amounts are required, and in some of the early studies on CBDs they were made radioactive with to increase sensitivity. The general technical problem of monitoring the binding of soluble, non-chromophoric ligands to soluble CBMs, has, however, been solved only in the last decade, with the advent of isothermal titration microcalorimetry... 

Leavitt, S., and Freire, E. (2001) Direct measurement of protein binding energetics by isothermal titration calorimetry. Curr. Opin. Struct. Biol. 11, 560-566. 

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