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

Abstract

The analysis of the salt dependence of protein-DNA complexes provides useful information about the non-specific electrostatic and sequence-specific parameters driving complex formation and stability. The differential scanning fluorimetry of GFP-tagged protein (DSF-GTP) assay has been geared with an automatic Tm peak recognition system and was applied for the high-throughput (HT) determination of salt-induced effects on the GFP-tagged DNA replication protein Tus in complex with various Ter and Ter-lock sequences. The system was designed to generate two-dimensional heat map profiles of Tus-GFP protein stability allowing for a comparative study of the effect of eight increasing salt concentrations on ten different Ter DNA species at once. The data obtained with the new HT DSF-GTP allowed precise dissection of the non-specific electrostatic and sequence-specific parameters driving Tus-Ter and Tus-Ter-lock complex formation and stability. The major factor increasing the thermal resistance of Tus-Ter-lock complexes in high-salt is the formation of the TT-lock, e.g. a 10-fold higher Kspe was obtained for Tus-GFP:Ter-lockB than for Tus-GFP:TerB. It is anticipated that the system can be easily adapted for the study of other protein-DNA complexes.

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.…”

Selective protein unfolding: a universal mechanism of action for the development of irreversible inhibitors

“…6,7 DSF and DSF-GTP are perfectly suited to screen compounds in high-throughput (HT) by measuring their effect on the thermal stability of a target protein. [8][9][10] In this work we screened an identical library of 940 compounds with four different proteins in order to identify target selective hits ( Fig. 1 and Fig.…”

“…T m were analyzed as previously described. 10 Structures of selected compounds and respective DT m are shown. See ESI, † for detailed methods and Fig.…”

“…Transition midpoint (T m ) values were recorded and processed as described previously using an automated T m peak recognition software. 10 For each compound, the net change in T m (DT m ) was determined by subtracting the T m of 'Target-GFP' from the T m of 'Target-GFP + compound' in identical buffer conditions. After the initial screen (801 compounds from Open Collection (Scaffolds) and 139 from Open Access Natural Product-Based Library, Compounds Australia) it became evident that of the compounds that affected a protein, the majority decreased the proteins' T m .…”