Rapid determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins

Abstract

The development of differential scanning fluorimetry and the high-throughput capability of Thermofluor have vastly facilitated the screening of crystallization conditions of proteins and large mutant libraries in structural genomics programs, as well as ligands in drug discovery and functional genomics programs. These techniques are limited by their requirement for both highly purified proteins and solvatochromic dyes, fueling the need for more robust technologies that can be used with crude protein samples. Here, we present the development of a new high-throughput technology for the quantitative determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins. This technology is based on the principle that a change in the proximal environment of GFP, such as unfolding and aggregation of the protein of interest, is measurable through its effect on the fluorescence of the fluorophore. Protein stability data was generated for twelve GFP-tagged proteins including monomeric and multimeric, DNA-binding, RNA-binding, proteolytic, heat-shock and metabolic proteins of Escherichia coli, Burkholderia pseudomallei, Staphylococcus aureus, dengue and influenza (H5N1) viruses. The technology is simple, fast and insensitive to variations in sample volumes, and the useful temperature and pH range is 30-80 uC and 5-11 respectively. The system does not require solvatochromic dyes, reducing the risk of interference. The protein samples are simply mixed with the test conditions in a 96-well plate and subjected to a melt-curve protocol using a real-time thermal cycler. The data are obtained within 1-2 h and include unique quality control measures.

Green fluorescent protein-based assays for high-throughput functional characterization and ligand-binding studies of biotin protein ligase

“…In both cases, the K obs were in agreement with the dissociation constants (K D ) obtained with alternative quantitative methods. 20,21 K obs is dened as the concentration of ligand at which the titration curve trendline for a given ligand (Fig. 3D) intercepts with log[ligand]-axis at the T m of the unliganded protein.…”

“…Recently, a new DSF platform based on the screening of GFP-tagged proteins (DSF-GTP) was developed to prole protein thermal stability in high-throughput (HT). [19][20][21] This new platform does not require the addition of a solvatochromic dye to the reaction thus eliminating the possibility of interferences with protein-ligand interactions. DSF-GTP has also the added advantage that it can be performed in the presence of additional proteins.…”

Dissecting the salt dependence of the Tus–Ter protein–DNA complexes by high-throughput differential scanning fluorimetry of a GFP-tagged Tus

“…Using a newly developed DSF-GTP assay we recently investigated the effect of salt on Tus-GFP:TerB and Tus-GFP:Ter-lockB complexes. 17 Nevertheless, the limited and scattered data we and others obtained on the effect of ionic strength on the Tus-Ter complex prompted us to further investigate this important system.…”

“…1A) to coordinate the termination of DNA replication opposite to oriC. [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] The Tus-Ter complex can only arrest a replisome approaching towards its non-permissive face through the formation of a Tus-Ter-lock (TT-lock), 18 whose action is to tighten the complex, avoiding dissociation of Tus. When the replisome approaches the permissive face of the complex, Tus rapidly dissociates from Ter.…”

“…In this study the effect of very low to high ionic strength on the stability of the ten primary Tus-Ter and their Tus-Ter-lock complexes was systematically analyzed using DSF-GTP 17 equipped with an automatic T m peak recognition system. The new automatic T m peak recognition system is able to automatically generate two-dimensional (2D) heat maps with an accuracy of 96% and greatly accelerates data analysis.…”