Microscale Thermophoresis
Encyclopedia
Microscale Thermophoresis (MST) is a technology for the analysis of biomolecule
s. 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 movement along the temperature gradient and is used to determine binding affinities
, binding kinetics
and activity kinetics. Events such as the phosphorylation
of a protein or the binding of small molecule
s to a target can be monitored. MST allows to measure interactions directly in solution without the need of an immobilization to a surface (immobilization-free technology). Microscale Thermophoresis was developed by the NanoTemper Technologies GmbH, a German high tech company with headquarters in Munich.
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 an 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 Fcold 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 Fhot. 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, ST determines the steady-state concentration ratio chot/ccold=exp(-ST ΔT) ≈ 1-ST ΔT under a temperature increase ΔT. The normalized fluorescence Fnorm=Fhot/Fcold measures mainly this concentration ratio, in addition to the temperature jump ∂F/∂T. In the linear approximation we find: Fnorm=1+(∂F/∂T-ST)ΔT. Due to the linearity of the fluorescence intensity and the thermophoretic depletion, the normalized fluorescence from the unbound molecule Fnorm(A) and the bound complex Fnorm(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: Fnorm=(1-x) Fnorm(A)+x Fnorm(AT).
Quantitative binding parameters are obtained by using a serial dilution of the binding substrate. By plotting Fnorm 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 KD as result.
Biomolecule
A biomolecule is any molecule that is produced by a living organism, including large polymeric molecules such as proteins, polysaccharides, lipids, and nucleic acids as well as small molecules such as primary metabolites, secondary metabolites, and natural products...
s. Microscale Thermophoresis
Thermophoresis
Thermophoresis, thermodiffusion, or Soret effect , is a phenomenon observed when a mixture of two or more types of motile particles are subjected to the force of a temperature gradient and the different types of particles respond to it differently. The term "Soret effect" normally means...
is the directed movement of particles in a microscopic temperature gradient
Temperature gradient
A temperature gradient is a physical quantity that describes in which direction and at what rate the temperature changes the most rapidly around a particular location. The temperature gradient is a dimensional quantity expressed in units of degrees per unit length...
. Any change of the hydration shell of biomolecules due to changes in their structure/conformation results in a relative change of movement along the temperature gradient and is used to determine binding affinities
Dissociation constant
In chemistry, biochemistry, and pharmacology, a dissociation constant is a specific type of equilibrium constant that measures the propensity of a larger object to separate reversibly into smaller components, as when a complex falls apart into its component molecules, or when a salt splits up into...
, binding kinetics
Receptor-ligand kinetics
In biochemistry, receptor-ligand kinetics is a branch of chemical kinetics in which the kinetic species are defined by different non-covalent bindings and/or conformations of the molecules involved, which are denoted as receptor and ligand....
and activity kinetics. Events such as the phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
of a protein or the binding of small molecule
Small molecule
In the fields of pharmacology and biochemistry, a small molecule is a low molecular weight organic compound which is by definition not a polymer...
s to a target can be monitored. MST allows to measure interactions directly in solution without the need of an immobilization to a surface (immobilization-free technology). Microscale Thermophoresis was developed by the NanoTemper Technologies GmbH, a German high tech company with headquarters in Munich.
Applications
Microscale Thermophoresis (MST) can be used to:- measure affinities between biomolecules including proteins, DNADNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
, RNARNARibonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
,peptides, molecule fragments, small molecules - measure interactions of biomolecules with nanoparticles, vesiclesVesicle (biology)A vesicle is a bubble of liquid within another liquid, a supramolecular assembly made up of many different molecules. More technically, a vesicle is a small membrane-enclosed sack that can store or transport substances. Vesicles can form naturally because of the properties of lipid membranes , or...
and nanodiscNanodiscA nanodisc is a synthetic model membrane system which assists in the study of membrane proteins. Nanodiscs are useful for membrane biology in the study of membrane proteins because they represent a more native environment than liposomes, detergent micelles or bicelles and allow the study of...
s - measure interactions with virusVirusA virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
es - measure the stoichiometryStoichiometryStoichiometry is a branch of chemistry that deals with the relative quantities of reactants and products in chemical reactions. In a balanced chemical reaction, the relations among quantities of reactants and products typically form a ratio of whole numbers...
of an interaction - measure enzyme kinetics
- measure methylationMethylationIn the chemical sciences, methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with, to be specific, a methyl group, rather than a larger carbon chain, replacing a hydrogen atom...
, phosphorylation and the like - characterize surface modifications of nanoparticles
- measure dissociation constants of multi-component reactions
- recognize different binding sites on a target molecule of interest
- measure the stability of biomolecules in blood, blood serum and blood plasma
- determine binding kinetics (koff/kon rates)
- study the adsorption of small molecules to lipid membranes or plasma proteins
- study protein aggregating compounds/conditions
- perform competition studies including the binding of substrates and inhibitors to an enzyme
Technology
Microscale thermophoresis (MST) is a new method that enables the quantitative analysis of molecular interactions in solution at the microliter scale. 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 ST: chot/ccold = exp(-ST ΔT)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 an 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
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
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 Fcold 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 Fhot. 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
Diffusion
Molecular diffusion, often called simply diffusion, is the thermal motion of all particles at temperatures above absolute zero. The rate of this movement is a function of temperature, viscosity of the fluid and the size of the particles...
D dictates the kinetics of depletion, ST determines the steady-state concentration ratio chot/ccold=exp(-ST ΔT) ≈ 1-ST ΔT under a temperature increase ΔT. The normalized fluorescence Fnorm=Fhot/Fcold measures mainly this concentration ratio, in addition to the temperature jump ∂F/∂T. In the linear approximation we find: Fnorm=1+(∂F/∂T-ST)ΔT. Due to the linearity of the fluorescence intensity and the thermophoretic depletion, the normalized fluorescence from the unbound molecule Fnorm(A) and the bound complex Fnorm(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: Fnorm=(1-x) Fnorm(A)+x Fnorm(AT).
Quantitative binding parameters are obtained by using a serial dilution of the binding substrate. By plotting Fnorm 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 KD as result.