Abstract

Differential scanning fluorimetry (DSF) is an inexpensive and quick assay used to assess the temperature-dependent unfolding of proteins. DSF uses an environmentally-responsive dye whose fluorescence is quenched in the presence of folded proteins. As samples are heated, the dye binds hydrophobic portions of the protein and fluorescence increases. Thus, the technique is used to determine the protein stability and melting temperature. Since ligands usually stabilize the proteins with which they interact, we can use DSF to determine their binding strengths by analyzing how the fluorescence curves shift upon binding.

We demonstrate the development and optimization of DSF assays for several different projects. One such project to be discussed utilized DSF to determine the potency of potential inhibitors on Dop and PafA, two enzymes involved in protein degradation in Mycobacterium tuberculosis, and this enabled the identification of non-antibiotic drug candidates for treating tuberculosis, highlighting one of many promising applications DSF.