The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability.
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.
Niesen
Frank H
FH
Structural Genomics Consortium, Botnar Research Centre, Oxford University, Oxford, UK. frank.niesen@sgc.ox.ac.uk
Berglund
Helena
H
Vedadi
Masoud
M
D000818:Animals; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D005456:Fluorescent Dyes / Q000032:analysis; D005470:Fluorometry / Q000379:methods*; D008024:Ligands; D062907:Oxaloacetic Acid / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013552:Swine / Q000378:metabolism; D013696:Temperature
D005456:Fluorescent Dyes; D008024:Ligands; D062907:Oxaloacetic Acid; D002950:Citrate (si)-Synthase