Thermofluor Assay: How to Make Your Protein Happy

Jana Škerlová

Institute of Organic Chemistry and Biochemistry, AS CR, v.v.i., Flemingovo nam. 2, Prague, Czech Republic

Thermofluor assay, also known as differential scanning fluorimetry or thermal shift assay, is a fluorescence-based biophysical method used to assess protein thermostability. Temperature gradient is applied on protein sample in the presence of a hydrophobic fluorescent dye, which binds to the hydrophobic core of the protein exposed during the unfolding process, and fluorescence signal is recorded. Melting temperature, determined from the protein melting curve, is a measure of protein stability in the given environment.

Thermofluor was originally developed for high-throughput screening for ligands [1], but the application of this versatile technique is not limited to drug discovery. It can be used for monitoring of protein-protein interactions [2], assessment of protein-ligand affinity [3], identification of protein function [4], evaluation of properties of protein constructs in engineering [5], or optimization of purification procedure [6].

Thermofluor assay is a key technique of structural biology [7]. Identification of buffer conditions or additives stabilizing the protein prior to crystallization greatly improves the hit rate in initial crystallization screening trials. Thermofluor-based buffer optimization can also give rise to alternative crystal forms with improved diffraction quality. Application of thermofluor will be demonstrated on example cases.

1. Pantoliano et al. (2001) High-density miniaturized thermal shift assays as a general strategy for drug discovery, J. Biomol. Screen. 6, 429-40.

2. Kopec & Schneider (2011) Comparison of fluorescence and light scattering based methods to assess formation and stability of protein-protein complexes, J. Struct. Biol. 175, 216-23.

3. Matulis et al. (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor, Biochemistry. 44, 5258-66.

4. Carver et al. (2005) Decrypting the biochemical function of an essential gene from Streptococcus pneumoniae using ThermoFluor technology, J. Biol. Chem. 280, 11704-12.

5. Lavinder et al. (2009) High-throughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering, J. Am. Chem. Soc. 131, 3794-5.