Microscale thermophoresis

Principle of the MST Technology: MST is performed in thin capillaries in free solution thus providing close-to-native conditions (immobilization free in any buffer, even in complex bioliquids) and a maintenance free instrument. When performing a MST experiment, a microscopic temperature gradient is induced by an infrared laser, and the directed movement of molecules is detected and quantified. Thermophoresis, the movement of the molecule in the temperature gradient, depends on three parameters that typically change upon interaction. Thus, the thermophoresis signal is plotted against the ligand concentration to obtain a dose-response curve, from which the binding affinity can be deduced.

Microscale thermophoresis (MST) is a technology for the interaction analysis of biomolecules. Microscale thermophoresis is the directed movement of particles in a microscopic temperature gradient. Any change of the hydration shell of biomolecules due to changes in their structure/conformation results in a relative change of the movement along the temperature gradient and is used to determine binding affinities. MST allows measurement of interactions directly in solution without the need of immobilization to a surface (immobilization-free technology).

Applications

Affinity

• between any kind of biomolecules including proteins, DNA, RNA, peptides, small molecules, fragments and ions
• for interactions with high molecular weight complexes, large molecule assemblies, even with liposomes, vesicles, nanodiscs, nanoparticles and viruses
• in any buffer, including serum and cell lysate
• in competition experiments (for example with subtrate and inhibitors)

Stoichiometry

Thermodynamic parameters

Additional information

• Sample property (homogeneity, aggregation, stability)
• Multiple binding sites, cooperativity

Technology

MST is based on the directed movement of molecules along temperature gradients, an effect termed thermophoresis. A spatial temperature difference ΔT leads to a depletion of molecule concentration in the region of elevated temperature, quantified by the Soret coefficient S_T: c_hot/c_cold = exp(-S_T ΔT)^[1][2]

Thermophoresis depends on the interface between molecule and solvent. Under constant buffer conditions, thermophoresis probes the size, charge and solvation entropy of the molecules. The thermophoresis of a fluorescently labeled molecule A typically differs significantly from the thermophoresis of a molecule-target complex AT due to size, charge and solvation entropy differences. This difference in the molecule's thermophoresis is used to quantify the binding in titration experiments under constant buffer conditions.

The thermophoretic movement of the fluorescently labelled molecule is measured by monitoring the fluorescence distribution F inside a capillary. The microscopic temperature gradient is generated by an IR-Laser, which is focused into the capillary and is strongly absorbed by water. The temperature of the aqueous solution in the laser spot is raised by up to ΔT=5 K. Before the IR-Laser is switched on a homogeneous fluorescence distribution F_cold is observed inside the capillary. When the IR-Laser is switched on, two effects, separated by their time-scales, contribute to the new fluorescence distribution F_hot. The thermal relaxation time is fast and induces a binding-dependent drop in the fluorescence of the dye due to its local environmental-dependent response to the temperature jump. On the slower diffusive time scale (10 s), the molecules move from the locally heated region to the outer cold regions. The local concentration of molecules decreases in the heated region until it reaches a steady-state distribution.

While the mass diffusion D dictates the kinetics of depletion, S_T determines the steady-state concentration ratio c_hot/c_cold=exp(-S_T ΔT) ≈ 1-S_T ΔT under a temperature increase ΔT. The normalized fluorescence F_norm=F_hot/F_cold measures mainly this concentration ratio, in addition to the temperature jump ∂F/∂T. In the linear approximation we find: F_norm=1+(∂F/∂T-S_T)ΔT. Due to the linearity of the fluorescence intensity and the thermophoretic depletion, the normalized fluorescence from the unbound molecule F_norm(A) and the bound complex F_norm(AT) superpose linearly. By denoting x the fraction of molecules bound to targets, the changing fluorescence signal during the titration of target T is given by: F_norm=(1-x) F_norm(A)+x F_norm(AT).^[3]

Quantitative binding parameters are obtained by using a serial dilution of the binding substrate. By plotting F_norm against the logarithm of the different concentrations of the dilution series, a sigmoidal binding curve is obtained. This binding curve can directly be fitted with the nonlinear solution of the law of mass action, with the dissociation constant K_D as result.^[4][5][6]

References

Further reading