A Brief Comparison of Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC)


  1.  MST

MST assay is based on thermophoresis, the directed movement of molecules in a temperature gradient induced by an infrared laser. Thermophoresis is highly sensitive to all types of binding-induced changes, such as size, charge, hydration shell or conformation, which allows for a precise quantification of molecular events. (Jerabek-Willemsen, André et al. 2014) Initial, the molecules are distributed homogenously with an initial fluorescence signal. When the IR laser is activated, the fluorescent signal is decreased as a ‘T-Jump’ form. With the turnoff of IR-laser, the molecule diffusion is back, solely driven by mass diffusion. The trace difference between a fluorescent molecule binding with or without non-fluorescent ligands indicates a binding signal. (Figure 1)

MST can handle weak conformation change on binding of two molecules in different buffer system, such as biological liquids. Another advantage is low sample consumption and MST can measure dissociation constants from pM to mM. (Wienken, Baaske et al. 2010, Jerabek-Willemsen, André et al. 2014) However, in most of cases, the sample should be labeled with hydrophobic fluorophores which would probably cause non-specific binding effects. (Table 1)

 Tina 1

Figure 1. MST setup and experiments. A. The machine Monilith NT. 115 from NanoTemper Technologies GmbH. B. Schematic representation of MST optics. C. Typical signal of a MST experiment. D. Typical binding experiment.(Jerabek-Willemsen, André et al. 2014)

  1. ITC

ITC is a biophysical technique to measure the heat exchange associated with molecular interactions at a constant temperature. (Duff Jr, Grubbs et al. 2011, Milev 2013) It directly determines the binding affinity (Ka), enthalpy changes (ΔH), and binding stoichiometry (n) of the interaction between two or more molecules in solution. The experimental methodology involves performing several titrant injections from a syringe (usually the ligand) into the solution (usually the macromolecule) in the cell, while maintaining the system at isobaric, quasi-isothermal conditions. When the ligands are injected to the cells, the ligands bind to macromolecules and the machine detects the heat upon binding. With several injections, ligands bound to protein continually. However, when the target protein becomes saturated with the ligand, less binding occurs and the heat change starts to decrease. If the macromolecule is saturated with ligand, no more binding occurs, and only heat of dilution is observed.

ITC has been widely applied as a major tool in drug discovery fields, validating and optimizing the hits (Leavitt and Freire 2001, Peters, Frasca et al. 2009) and also in binding studies, such as protein-protein, protein-DNA, small molecule-protein interactions(de Azevedo, Walter et al. 2008, Liang 2008). ITC is a fast and straight way to detect binding affinity of two molecules by the change of binding enthalpy. However, some complexes may exhibit rather small binding enthalpies that are not suitable for the ITC measurement. (Table 1)

MST ITC
Advantages ²   Small sample size

²   Immobilization free

²   Minimal contamination of the sample

²   Ability to measure complex mixtures

²   Wide size range for interactants (ions to MDa complexes)

²   Ability to determine thermodynamic binding parameters in a single experiment

²   Modification of binding partners are not required

 

Disadvantages ²   Hydrophobic fluorescent labelling required, may cause non-specific binding

²   No kinetic information

²   Highly sensitive to any change in molecular properties

²   Large sample quantity needed

²   Kinetics cannot be determined

²   Limited range for consistently measured binding affinities

²   Non-covalent complexes may exhibit rather small binding enthalpies since signal is proportional to the binding enthalpy

²   Not suitable for HTS

Table 1. Advantages and disadvantages of ITC and MST.

  1. Ligand-protein binding affinities detected by ITC and MST

One example is about comparing the biophysical data of small molecules with Protein kinase CK2 using both MST and ITC assays.(Winiewska, Bugajska et al. 2017) In this paper, the interactions of four halogenated benzotriazoles with the catalytic subunit of human protein kinase CK2 had been investigated. Among the four compounds, only one compound (5-BrBt) had a consistent binding affinity data in both MST and ITC assays, the solubility of which substantially exceeded the ligand concentration. For another three compounds, when the compounds titrated to the protein solution for ITC measurement, the binding affinities determined by ITC were around 10-folded weaker than by MST. The main problem was the limited titrant solubility that resulted in the formation of nano-aggregates. The issue was ignored by titrating the protein to the compound solution as the protein was soluble enough. (Figure 2) The protein-ligand affinities that derived from ITC may be underestimated because of the compound solubility problem, while the problem can be avoided by MST. Tina 2

Figure 2. Correlation between MST- and ITC-derived binding affinities determined for complexes of halogenated benzotriazoles with hCK2α. Kd(ITC), were obtained with ITC experiment, in which either inhibitor (red) or protein (blue) was used as a titrant. Vertical and horizontal bars represent standard deviation (MST) and 67% confidence intervals (ITC), respectively.(Winiewska, Bugajska et al. 2017)

Blog written by Xiangrong (Tina) Chen

de Azevedo, J., F. Walter and R. Dias (2008). “Experimental approaches to evaluate the thermodynamics of protein-drug interactions.” Current drug targets 9(12): 1071-1076.

Duff Jr, M. R., J. Grubbs and E. E. Howell (2011). “Isothermal titration calorimetry for measuring macromolecule-ligand affinity.” J Vis Exp 55: e2796.

Jerabek-Willemsen, M., T. André, R. Wanner, H. M. Roth, S. Duhr, P. Baaske and D. Breitsprecher (2014). “MicroScale Thermophoresis: Interaction analysis and beyond.” Journal of Molecular Structure 1077: 101-113.

Leavitt, S. and E. Freire (2001). “Direct measurement of protein binding energetics by isothermal titration calorimetry.” Current opinion in structural biology 11(5): 560-566.

Liang, Y. (2008). “Applications of isothermal titration calorimetry in protein science.” Acta biochimica et biophysica Sinica 40(7): 565-576.

Milev, S. (2013). “Isothermal titration calorimetry: Principles and experimental design.” General Electric 9.

Peters, W. B., V. Frasca and R. K. Brown (2009). “Recent developments in isothermal titration calorimetry label free screening.” Combinatorial chemistry & high throughput screening 12(8): 772-790.

Wienken, C. J., P. Baaske, U. Rothbauer, D. Braun and S. Duhr (2010). “Protein-binding assays in biological liquids using microscale thermophoresis.” Nature communications 1: ncomms1093.

Winiewska, M., E. Bugajska and J. Poznański (2017). “ITC-derived binding affinity may be biased due to titrant (nano)-aggregation. Binding of halogenated benzotriazoles to the catalytic domain of human protein kinase CK2.” PloS one 12(3): e0173260.

 

 

 

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