Thermal Shift Assay

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Principle of the thermal shift assay

A thermal shift assay (TSA, synonym thermal fluoroassay or differential scanning Fluorimetry , DSF) is a biochemical method the change to measure the thermal stability of the folding of a protein by adding a binding partner . It is therefore a method for determining protein-protein , protein-DNA and protein-lipid interactions as well as interactions between proteins and low molecular weight compounds .

principle

The folding of a protein is often stabilized by binding another molecule. The stabilization is expressed, among other things, by an increased thermal stability of the protein, in which the biological activity of the protein is retained even at comparatively higher temperatures and denaturation only begins at higher temperatures.

The thermal shift assay is carried out by adding different dilution levels of a binding partner. Two measurement methods have been implemented. On the one hand, an indirect method by adding a fluorescent protein dye such. B. SYPRO Orange or 1-anilinonaphthalene-8-sulfonic acid (ANS). The test batches of the dilution levels and a negative control are incubated in a real-time cycler , which measures the fluorescence in the test batches with increasing temperature. On the other hand, the direct measurement of intrinsic fluorescence , self-fluorescence, tryptophans (and, to a lesser extent, tyrosines ), which due to their polarity are primarily located inside a protein structure. A binding event usually has an effect on the structure or folding of the protein, which can result in a change in the positions of the tryptophans or tyrosines within the structure. Such a change in position causes a change in fluorescence, which is due to the change in environmental conditions ( hydration , electron density ). This effect becomes particularly clear when a tryptophan or tyrosine is involved in the binding event. In many cases, this provides information on structural changes during a bonding process in addition to thermal stabilization.

A variant of the method called CETSA ( cellular thermal shift assay ) uses cell cultures instead of purified proteins, whereby the first part of the experiment, the binding of a binding partner to a protein, can take place in vivo . The buffers used in denaturation affect the change in the thermal stability of a protein. Due to the comparatively low cost of materials, thermal shift assays are suitable for measurements in high throughput .

Applications

Thermal shift assays are used, among other things, in protein characterization , protein engineering and drug design.

Thermal shift assays can also be used to find suitable buffer conditions for a protein.

A related method to identify optimal buffer compositions for a target protein or biopharmaceutical is nanoDSF . nanoDSF measures the stability of protein folding in thermal and chemical unfolding experiments. Changes in the intrinsic fluorescence of tryptophan during unfolding are detected.

Individual evidence

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  5. ^ Daniel E. Koshland : Application of a Theory of Enzyme Specificity to Protein Synthesis. In: Proceedings of the National Academy of Sciences . Volume 44, Number 2, February 1958, ISSN  0027-8424 , pp. 98-104, PMID 16590179 , PMC 335371 (free full text).
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  8. ↑ Anders O. Magnusson, Anna Szekrenyi, Henk-Jan Joosten, James Finnigan, Simon Charnock: nanoDSF as screening tool for enzyme libraries and biotechnology development . In: The FEBS Journal . tape 286 , no. 1 , 2019, ISSN  1742-4658 , p. 184-204 , doi : 10.1111 / febs.14696 .
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  10. ^ R. Jafari, H. Almqvist, H. Axelsson, M. Ignatushchenko, T. Lundbäck, P. Nordlund, D. Martinez Molina: The cellular thermal shift assay for evaluating drug target interactions in cells. In: Nature protocols. Volume 9, number 9, September 2014, ISSN  1750-2799 , pp. 2100-2122, doi : 10.1038 / nprot.2014.138 , PMID 25101824 .
  11. ↑ L. Reinhard, H. Mayerhofer, A. Geerlof, J. Mueller-Dieckmann, MS Weiss: Optimization of protein buffer cocktails using Thermofluor. In: Acta crystallographica. Section F, Structural biology and crystallization communications. Volume 69, Pt 2 February 2013, ISSN  1744-3091 , pp. 209-214, doi : 10.1107 / S1744309112051858 , PMID 23385769 , PMC 3564630 (free full text).
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