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Application of temperature-responsive HIS-tag fluorophores to differential scanning fluorimetry screening of small molecule libraries

Affiliation
National Center for Advancing Translational Sciences ,National Institutes of Health ,Rockville ,MD ,United States
Ronzetti, Michael H.;
Affiliation
National Center for Advancing Translational Sciences ,National Institutes of Health ,Rockville ,MD ,United States
Baljinnyam, Bolormaa;
Affiliation
National Center for Advancing Translational Sciences ,National Institutes of Health ,Rockville ,MD ,United States
Itkin, Zina;
Affiliation
National Center for Advancing Translational Sciences ,National Institutes of Health ,Rockville ,MD ,United States
Jain, Sankalp;
Affiliation
National Center for Advancing Translational Sciences ,National Institutes of Health ,Rockville ,MD ,United States
Rai, Ganesha;
Affiliation
National Center for Advancing Translational Sciences ,National Institutes of Health ,Rockville ,MD ,United States
Zakharov, Alexey V.;
Affiliation
Department of Veterinary Medicine ,College of Agriculture and Natural Resources ,University of Maryland ,College Park ,MD ,United States
Pal, Utpal;
Affiliation
National Center for Advancing Translational Sciences ,National Institutes of Health ,Rockville ,MD ,United States
Simeonov, Anton

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 histidine-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 HIS-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 T m values with an average coefficient of variation of 0.51% and median coefficient of variation of compound T m 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.

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License Holder: Copyright © 2022 Ronzetti, Baljinnyam, Itkin, Jain, Rai, Zakharov, Pal and Simeonov.

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