title: Determination of protein-ligand interactions using differential scanning fluorimetry.
abstract: A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.
authors.affiliation: Department of Biosciences, University of Exeter.
authors.forename: Mirella
authors.initials: M
authors.lastname: Vivoli
authors.affiliation: Department of Biosciences, University of Exeter.
authors.forename: Halina R
authors.initials: HR
authors.lastname: Novak
authors.affiliation: Department of Biosciences, University of Exeter.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Littlechild
authors.affiliation: Department of Biosciences, University of Exeter; N.J.Harmer@exeter.ac.uk.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Harmer
mesh_terms: D005470:Fluorometry / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods; D007700:Kinetics; D008024:Ligands; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008024:Ligands; D011506:Proteins; D054852:Small Molecule Libraries