H-033. Evaluation of a Fluorescence-Based Thermal Shift Assay to Map Ligands with Binding Proteins

S. E. Giuliani, A. M. Frank, L. J. Field, J. L. Saunders, F. R. Collart;
Argonne Natl. Lab., Argonne, IL.

All cells contain proteins that sense the environment and mediate transport and signaling events that lead to changes in metabolism and/or initiate changes in gene expression at the level of transcription. Mapping of ligands with these binding/sensor proteins is critical to our understanding of cell biochemistry and is essential for modeling cellular processes and the rational design of engineered organisms. We have scanned the Protein Data Bank (PDB) and identified more than 25 structure categories with unique combinations of sensor type proteins and ligands. The bound ligands can be grouped into several categories such as amino acids, metals, small ions, sugars, and vitamins. We have used these reference PDB sequences to identify potential homologs in a set of reagent bacterial genomes as candidates for functional screening using a library of candidate binding ligands. A fluorescence-based thermal shift assay is used for identification of bound ligands. Carbonic Anhydrase I was used as a positive control test protein to establish assay parameters and illustrate the suitability of this approach for ligand binding studies. The native form of this protein displays a concentration-dependent thermal shift and the addition of a tight binding inhibitor results in an increase in the Tm indicating enhanced protein stability. This assay has been extended to evaluate a panel of twelve target proteins with a small ligand library. In this preliminary study, we focused on structures that contain native ligands, to enable development of an initial ligand library. The present focus is on periplasmic binding proteins but there are no restrictions to extending this type of analysis to other types of ligands and binding proteins. The set of control proteins has been purified and characterized by CD spectroscopy. These targets will enable evaluation of this assay as a method to improve gene/protein functional assignments of sensor type proteins by developing high throughput methods to match ligands with their binding proteins.

Acknowledgement

The submitted abstract has been created by UChicago Argonne, LLC, Operator of Argonne National Laboratory (“Argonne”). Argonne, a U.S. Department of Energy Office of Science laboratory, is operated under Contract No. DE-AC02-06CH11357