Abstract
A standard operating procedure for a fluorescence-based thermal shift assay (FTSA) is provided describing its typical applications, advantages and limitations. FTSA is a simple, robust, universal and quick assay to determine protein–ligand binding affinities and protein stabilities in the presence of various excipients and solution conditions. Therefore, the assay is very useful for the straightforward characterization of new recombinantly produced proteins. The assay has a wide dynamic range enabling simultaneous determination of affinities in the milimolar to picomolar range. The assay could be used for essentially any protein that is sufficiently soluble and stable in the studied aqueous solution. Here we provide examples and typical experimental protocols for both affinity and stability determinations.
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References
Anderson SR, Weber G (1969) Fluorescence polarization of the complexes of 1-anilino-8-naphthalenesulfonate with bovine serum albumin. Evidence for preferential orientation of the ligand. Biochemistry 8:371–377
Baranauskienė L, Hilvo M, Matulienė J et al (2010) Inhibition and binding studies of carbonic anhydrase isozymes I, II and IX with benzimidazo[1,2-c][1,2,3]thiadiazole-7-sulphonamides. J Enzyme Inhib Med Chem 25:863–870. https://doi.org/10.3109/14756360903571685
Biter AB, de la Peña AH, Thapar R et al (2016) DSF guided refolding as a novel method of protein production. Sci Rep 6:18906. https://doi.org/10.1038/srep18906
Brandts JF, Lin LN (1990) Study of strong to ultratight protein interactions using differential scanning calorimetry. Biochemistry 29:6927–6940
Cimmperman P, Matulis D (2011) Protein thermal denaturation measurements via a fluorescent dye. In: Podjarny A, Dejaegere AP, Kieffer B (eds) RSC biomolecular sciences. Royal Society of Chemistry, Cambridge, pp 247–274
Cimmperman P, Baranauskienė L, Jachimovičiūtė S et al (2008) A quantitative model of thermal stabilization and destabilization of proteins by ligands. Biophys J 95:3222–3231. https://doi.org/10.1529/biophysj.108.134973
Crowther GJ, He P, Rodenbough PP et al (2010) Use of thermal melt curves to assess the quality of enzyme preparations. Anal Biochem 399:268–275. https://doi.org/10.1016/j.ab.2009.12.018
Gao K, Oerlemans R, Groves MR (2020) Theory and applications of differential scanning fluorimetry in early-stage drug discovery. Biophys Rev 12:85–104. https://doi.org/10.1007/s12551-020-00619-2
Kairys V, Baranauskiene L, Kazlauskiene M et al (2019) Binding affinity in drug design: experimental and computational techniques. Expert Opin Drug Discov. https://doi.org/10.1080/17460441.2019.1623202
Layton CJ, Hellinga HW (2010) Thermodynamic analysis of ligand-induced changes in protein thermal unfolding applied to high-throughput determination of ligand affinities with extrinsic fluorescent dyes. Biochemistry 49:10831–10841. https://doi.org/10.1021/bi101414z
Layton CJ, Hellinga HW (2011) Quantitation of protein–protein interactions by thermal stability shift analysis. Protein Sci 20:1439–1450. https://doi.org/10.1002/pro.674
Linkuvienė V, Zubrienė A, Manakova E et al (2018) Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design. Q Rev Biophys. https://doi.org/10.1017/S0033583518000082
Lo M-C, Aulabaugh A, Jin G et al (2004) Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery. Anal Biochem 332:153–159. https://doi.org/10.1016/j.ab.2004.04.031
Matulis D, Lovrien R (1998) 1-anilino-8-naphthalene sulfonate anion-protein binding depends primarily on ion pair formation. Biophys J 74:422–429. https://doi.org/10.1016/S0006-3495(98)77799-9
Matulis D, Baumann CG, Bloomfield VA, Lovrien RE (1999) 1-Anilino-8-naphthalene sulfonate as a protein conformational tightening agent. Biopolymers 49:451–458. https://doi.org/10.1002/(SICI)1097-0282(199905)49:6%3c451::AID-BIP3%3e3.0.CO;2-6
Matulis D, Kranz JK, Salemme FR, Todd MJ (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44:5258–5266. https://doi.org/10.1021/bi048135v
McDonnell PA, Yanchunas J, Newitt JA et al (2009) Assessing compound binding to the Eg5 motor domain using a thermal shift assay. Anal Biochem 392:59–69. https://doi.org/10.1016/j.ab.2009.05.044
Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Prot 2:2212–2221. https://doi.org/10.1038/nprot.2007.321
Pantoliano MW, Petrella EC, Kwasnoski JD et al (2001) High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen 6:429–440. https://doi.org/10.1177/108705710100600609
Petrauskas V, Zubrienė A, Todd MJ, Matulis D (2019) Inhibitor binding to carbonic anhydrases by fluorescent thermal shift assay. In: Matulis D (ed) Carbonic anhydrase as drug target: thermodynamics and structure of inhibitor binding. Springer International Publishing, Cham, pp 63–78
Scott DE, Spry C, Abell C (2016) Differential scanning fluorimetry as part of a biophysical screening cascade. In: Erlanson DA, Jahnke W (eds) Methods and principles in medicinal chemistry. Wiley-VCH Verlag GmbH & Co KGaA, Weinheim, pp 139–172
Semisotnov GV, Rodionova NA, Razgulyaev OI et al (1991) Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers 31:119–128
Slavik J, Horak J, Rihova L, Kotyk A (1982) Anilinonaphthalene sulfonate fluorescence and amino acid transport in yeast. J Membr Biol 64:175–179
Vedadi M, Niesen FH, Allali-Hassani A et al (2006) Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc Natl Acad Sci 103:15835–15840. https://doi.org/10.1073/pnas.0605224103
Wang Y, van Oosterwijk N, Ali AM et al (2017) A systematic protein refolding screen method using the DGR approach reveals that time and secondary TSA are essential variables. Sci Rep 7:9355. https://doi.org/10.1038/s41598-017-09687-z
Yanchunas J, Langley DR, Tao L et al (2005) Molecular basis for increased susceptibility of isolates with atazanavir resistance-conferring substitution I50L to other protease inhibitors. Antimicrob Agents Chemother 49:3825–3832. https://doi.org/10.1128/AAC.49.9.3825-3832.2005
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Authors acknowledge the ARBRE network and the COST project MOBIEU CA15126.
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Special Issue: COST Action CA15126, MOBIEU: between atom and cell.
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Kazlauskas, E., Petrauskas, V., Paketurytė, V. et al. Standard operating procedure for fluorescent thermal shift assay (FTSA) for determination of protein–ligand binding and protein stability. Eur Biophys J 50, 373–379 (2021). https://doi.org/10.1007/s00249-021-01537-1
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DOI: https://doi.org/10.1007/s00249-021-01537-1