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

High-throughput differential scanning fluorimetry of GFP-tagged proteins (HT-DSF-GTP) was applied for the identification of novel enzyme inhibitors acting by a mechanism termed: selective protein unfolding (SPU). Four different protein targets were interrogated with the same library to identify target-selective hits. Several hits selectively destabilized bacterial biotin protein ligase. Structure–activity relationship data confirmed a structure-dependent mechanism of protein unfolding. Simvastatin and altenusin were confirmed to irreversibly inactivate biotin protein ligase. The principle of SPU combined with HT-DSF-GTP affords an invaluable and innovative workflow for the identification of new inhibitors with potential applications as antimicrobials and other biocides

Delineation of the ancestral tus-dependent replication fork trap

In Escherichia coli, DNA replication termination is orchestrated by two clusters of Ter sites forming a DNA replication fork trap when bound by Tus proteins. The formation of a ‘locked’ Tus– Ter complex is essential for halting incoming DNA replication forks. However, the absence of replication fork arrest at some Ter sites raised questions about their significance. In this study, we examined the genome-wide distribution of Tus and found that only the six innermost Ter sites (TerA–E and G) were significantly bound by Tus. We also found that a single ectopic insertion of TerB in its non-permissive orientation could not be achieved, advocating against a need for ‘back-up’ Ter sites. Finally, examination of the genomes of a variety of Enterobacterales revealed a new replication fork trap architecture mostly found outside the Enterobacteriaceae family. Taken together, our data enabled the delineation of a narrow ancestral Tus-dependent DNA replication fork trap consisting of only two Ter sites

Differential Tus–Ter binding and lock formation: implications for DNA replication termination in Escherichia coli

In E. coli, DNA replication termination occurs at Ter sites and is mediated by Tus. Two clusters of five Ter sites are located on each side of the terminus region and constrain replication forks in a polar manner. The polarity is due to the formation of the Tus–Ter-lock intermediate. Recently, it has been shown that DnaB helicase which unwinds DNA at the replication fork is preferentially stopped at the non-permissive face of a Tus–Ter complex without formation of the Tus–Ter-lock and that fork pausing efficiency is sequence dependent, raising two essential questions: Does the affinity of Tus for the different Ter sites correlate with fork pausing efficiency? Is formation of the Tus–Ter-lock the key factor in fork pausing? The combined use of surface plasmon resonance and GFP-Basta showed that Tus binds strongly to TerA–E and G, moderately to TerH–J and weakly to TerF. Out of these ten Ter sites only two, TerF and H, were not able to form significant Tus–Ter-locks. Finally, Tus's resistance to dissociation from Ter sites and the strength of the Tus–Ter-locks correlate with the differences in fork pausing efficiency observed for the different Ter sites by Duggin and Bell (2009)

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

The analysis of the salt dependence of protein-DNA complexes provides useful information about the nonspecific 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 T-m 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 K-spe 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

A polyplex qPCR-based binding assay for protein–DNA interactions

The measurement of protein–DNA interactions is difficult and often involves radioisotope-labelled DNA to obtain the desired assay sensitivity. More recently, high-throughput proteomic approaches were developed but they generally lack sensitivity. For these methods, the level of technical difficulties involved is high due to the need for specialised facilities or equipment and training. The new qPCR-based DNA-binding assay involves immunoprecipitation of a GFP-tagged DNA-binding protein in complex with various DNA targets (Ter sites) followed by qPCR quantification, affording a very sensitive and quantitative method that can be performed in polyplex. Using a single binding reaction, the binding specificity of the DNA replication terminator protein Tus for ten termination sites TerA–J could be obtained for the first time in just a few hours. This new qPCR DNA-binding assay can easily be adapted to determine the binding specificity of virtually any soluble and functional epitope-tagged DNA-binding protein

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

The analysis of the salt dependence of protein-DNA complexes provides useful information about the nonspecific 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 T-m 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 K-spe 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

Dickensian

Investigations into the photocrosslinking kinetics of the protein Tus with various bromodeoxyuridine-substituted Ter DNA variants highlight the potential use of this complex as a photoactivatable connector between proteins of interest and specific DNA sequences

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

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 °C 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