Application of Temperature-Responsive HIS-Tag Fluorophores to Differential Scanning Fluorimetry Screening of Small Molecule Libraries

Abstract

Differential scanning fluorimetry is a rapid and economical biophysical technique used to monitor perturbations to protein structure during a thermal gradient most often by detecting protein unfolding events through an environment-sensitive fluorophore. By employing an NTA-complexed fluorophore that is sensitive to nearby structural changes in 6xHIS-tagged protein, a robust and sensitive differential scanning fluorimetry (DSF) assay is established with the specificity of an affinity tag-based system. We developed, optimized, and miniaturized this histidine-tag DSF assay (HIS-DSF) into a 1536-well high-throughput biophysical platform using the Borrelial high temperature requirement A protease (BbHtrA) as a proof of concept for the workflow. A production run of the BbHtrA HIS-DSF assay showed a tight negative control group distribution of Tm values with an average coefficient of variation of 0.51% and median coefficient of variation of compound Tm of 0.26%. The HIS-DSF platform will provide an additional assay platform for future drug discovery campaigns with applications in buffer screening and optimization, target engagement screening, and other biophysical assay efforts.