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The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability

Abstract

Differential scanning fluorimetry (DSF) is a rapid and inexpensive screening method to identify low-molecular-weight ligands that bind and stabilize purified proteins. The temperature at which a protein unfolds is measured by an increase in the fluorescence of a dye with affinity for hydrophobic parts of the protein, which are exposed as the protein unfolds. A simple fitting procedure allows quick calculation of the transition midpoint; the difference in the temperature of this midpoint in the presence and absence of ligand is related to the binding affinity of the small molecule, which can be a low-molecular-weight compound, a peptide or a nucleic acid. DSF is best performed using a conventional real-time PCR instrument. Ligand solutions from a storage plate are added to a solution of protein and dye, distributed into the wells of the PCR plate and fluorescence intensity measured as the temperature is raised gradually. Results can be obtained in a single day.

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Figure 1: Optical properties and fluorescence signal in the presence of lysozyme (native versus denatured) for selected dyes that can be used for DSF.
Figure 2: Typical recording of fluorescence intensity versus temperature for the unfolding of protein (citrate synthase) in the presence of SYPRO orange.
Figure 3: Optimum excitation of SYPRO orange in protein buffer and gain in fluorescence upon protein unfolding.
Figure 4: Layout for a 96-well plate, enabling screening of sets of 29 compounds under three different conditions in parallel.
Figure 5: Concentration-dependent stabilization of citrate synthase by oxaloacetate.
Figure 6: Visualization of the screen results for citrate synthase against physiologically relevant compounds.

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Acknowledgements

We are grateful to Oleg Y. Fedorov and Guillermo Senisterra for fruitful discussions, and to Aled M. Edwards and Patrick J. Finerty Jr. for critically reading the manuscript. The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co. Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust.

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Correspondence to Frank H Niesen.

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Niesen, F., Berglund, H. & Vedadi, M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2, 2212–2221 (2007). https://doi.org/10.1038/nprot.2007.321

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