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SPS - Saclay Plant Sciences

Thermophoresis: an innovative method for biomolecular interaction analysis

A MicroScaleThermophoresis (MST) device, co-funded by the SPS LabEx, AgroParisTech, the “Ile de France” region and the Jean-Pierre Bourgin Institute (IJPB), has been acquired in 2015, to equip the Plant Observatory Biochemistry platform of the IJPB at the INRA site of Versailles. Through the Plant Observatory, the MST technique is available to all the scientists of the SPS LabEx, the research/teaching community of the Université Paris-Saclay and all our academic partners.

Thermophoresis, or Soret effect, measures the directed motion of molecules in a temperature gradient. The movement of a compound in solution depends on various molecular properties such as size, charge, and hydration shell according to the Soret law. When two molecules interact, at least one of these parameters is modified, resulting in a modification of the thermophoresis curve.

Based on the thermophoresis principle, MST is a cutting-edge and easy to handle technology developed to study and quantify the interactions between diverse biomolecules, purified or not.

The equipment we acquired makes it possible to detect variations in the thermophoretic mobility of a fluorescent molecule (naturally or by fluorescent labeling) subjected to a micro gradient of temperature established in a capillary, in the presence of increasing concentrations of a specific interacting agent. Exploiting the thermophoretic properties of a compound for molecular interaction studies is an innovative approach.

Gonneau1

https://doi.org/10.1016/j.molstruc.2014.03.009

(A) MST equipment (Monolith NT.115 from NanoTemper Technologies GmbH). (B) Schematic representation of MST optics. The fluorescence within the capillaries (16 capillaries of ∼4 μL) is excited and measured through the same optical device. A focused IR-Laser heats locally and the thermophoresis of fluorescent molecules through the temperature gradient is detected (C) Initially, the molecules are homogeneously distributed and a constant “initial fluorescence” is recorded. After activation of the IR laser, the thermophoretic movement of the fluorescencently labeled molecules out of the heated sample volume can be measured. After deactivation of the IR-Laser, a “backdiffusion” of molecules occurs, solely driven by mass diffusion. (D) Typical binding experiment. The thermophoretic movement of a fluorescent molecule (black trace; “unbound”) changes upon binding to a non-fluorescent ligand (red trace; “bound”), resulting in different traces. Titration of the non-fluorescent ligand results in a gradual change in thermophoresis, which is plotted as ΔFnorm to generate a binding curve, which can be fitted to derive binding constants.

Beyond its simplicity of implementation, the MST technique has many advantages, including:

  • Sensitivity: the method is based on the recording of minimal variations in mass, charge or solvation. It allows the measurement of interactions with essentially no limitation on molecule size.
  • Measurements in solution: contrary to other techniques of interaction studies, no immobilization is required and therefore no conformational disturbance is to be feared.
  • Very wide application ranges: protein / protein, protein / ligand, protein / sugar, protein / DNA, ligand / viral particle, bacteria, cell, etc.
  • Fluorescent labeling of a purified protein: the labeling, either on lysines or on cysteines contained in the protein, can be achieved in less than one hour.
  • Purification of the molecules not always required: for example, GFP-fusion proteins can be analyzed directly in cell extracts without purification, which is interesting if cellular partners are needed for the interaction  (partner protein, cofactor, etc.)
  • Possibility to use the fluorescence of different molecules, labeled nucleic acids or oligosaccharides, for example.
  • Use of low amount of the labeled species: 10 μl of a pM to nM solution, with a large dynamic range of dissociation constants (KD 10-11 to 10-2 M).
  • Rapidity: different experimental conditions can be tested quickly, such as pH, in the presence of salts, detergents etc., or the type of capillary (standard or substituted). Once the experimental conditions are established, the dissociation constants can be obtained in 10 min.
  • Relatively low cost consumables: about 300 € for 1000 standard capillaries.
  • Intuitive data acquisition and processing software.

MST requires rigor and precision, particularly with regard to the distribution of the fluorescent partner, but its simplicity of use nevertheless makes it possible to propose this method of interaction study on a technical platform after a short training. MST can also allow students to understand the notion of specific interaction and to experimentally measure dissociation constants.

Since the acquisition of the MST equipment, IJPB scientists have obtained encouraging results in various research programs and others are being published. Interactions between receptors and peptide ligands, or receptor and hormone, or between a transcription factor and its DNA target have been highlighted.

The Plant Observatory welcomes SPS LabEx scientists in order to help them become familiar with this technique, to initiate preliminary assays together and to study if MST is a relevant technique for the potential molecular interactions studied.

Gonneau2

References

Reference article :

Wienken CJ, Baaske P, Rothbauer U, Braun D, Duhr S. Protein-binding assays in biological liquids using microscale thermophoresis. Nat Commun. 2010 Oct 19;1:100. doi: 10.1038/ncomms1093. PMID: 20981028

Protein/protein interaction:

Chen ST, He NY, Chen JH, Guo FQ. Identification of core subunits of photosystem II as action sites of HSP21, which is activated by the GUN5-mediated retrograde pathway in Arabidopsis. Plant J. 2017 89(6):1106-1118. doi:10.1111/tpj.13447. Epub 2017 Feb 27. PubMed PMID: 27943531.

Sénéchal F, L'Enfant M, Domon JM, Rosiau E, Crépeau MJ, Surcouf O, Esquivel-Rodriguez J, Marcelo P, Mareck A, Guérineau F, Kim HR, Mravec J, Bonnin E, Jamet E, Kihara D, Lerouge P, Ralet MC, Pelloux J, Rayon C. Tuning of Pectin Methylesterification: Pectin methylesterase inhibitor 7 modulates the processive activity of co-expressed pectin methylesterase 3 in a PH-dependent manner. J Biol Chem. 2015 290(38):23320-35. doi: 10.1074/jbc.M115.639534. Epub 2015 Jul16. PubMed PMID: 26183897; PubMed Central PMCID: PMC4645611.

Protein / small organic compound interaction:

Zhang W, Li X, Zhang G, Ding Y, Ran L, Luo L, Wu J, Hu D, Song B. Binding interactions between enantiomeric α-aminophosphonate derivatives and tobacco mosaic virus coat protein. Int J Biol Macromol. 2017 Jan;94(Pt A):603-610. doi: 10.1016/j.ijbiomac.2016.10.027. Epub 2016 Oct 13. PubMed PMID: 27746356.

Protein / nucleic acid interaction:

Girke C, Arutyunova E, Syed M, Traub M, Möhlmann T, Lemieux MJ. High yield expression and purification of equilibrative nucleoside transporter 7 (ENT7) from Arabidopsis thaliana. Biochim Biophys Acta. 2015 1850(9):1921-9. doi: 10.1016/j.bbagen.2015.06.003. Epub 2015 Jun 12. PubMed PMID: 26080001.

Protein / ion interaction:

Parker JL, Newstead S. Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1. Nature. 2014 507(7490):68-72. doi: 10.1038/nature13116. Epub 2014 Feb 26. PMID: 24572366