Differences in Unfolding Energetics of CcdB Toxins From V. fischeri and E. coli

Ccd system is a toxin-antitoxin module (operon) located on plasmids and chromosomes of bacteria. CcdB(F) encoded by ccd operon located on Escherichia coli plasmid F and CcdB(Vfi) encoded by ccd operon located on Vibrio fischeri chromosome are members of the CcdB family of toxins. Native CcdBs are dimers that bind to gyrase-DNA complexes and inhibit DNA transcription and replication. While thermodynamic stability and unfolding characteristics of the plasmidic CcdB(F) in denaturant solutions are reported in detail, the corresponding information on the chromosomal CcdB(Vfi) is rather scarce. Therefore, we studied urea-induced unfolding of CcdB(Vfi) at various temperatures and protein concentrations by circular dichroism spectroscopy. Global model analysis of spectroscopic data suggests that CcdB(Vfi) dimer unfolds to the corresponding monomeric components in a reversible two-state manner. Results reveal that at physiological temperatures CcdB(Vfi) exhibits lower thermodynamic stability compared to CcdB(F). At high urea concentrations CcdB(Vfi), similarly to CcdB(F), retains a significant amount of secondary structure. Differences in thermodynamic parameters of CcdB(Vfi) and CcdB(F) unfolding can reasonably be explained by the differences in their structural features

Differences in Unfolding Energetics of CcdB Toxins From V. fischeri and E. coli

Ccd system is a toxin-antitoxin module (operon) located on plasmids and chromosomes of bacteria. CcdB(F) encoded by ccd operon located on Escherichia coli plasmid F and CcdB(Vfi) encoded by ccd operon located on Vibrio fischeri chromosome are members of the CcdB family of toxins. Native CcdBs are dimers that bind to gyrase-DNA complexes and inhibit DNA transcription and replication. While thermodynamic stability and unfolding characteristics of the plasmidic CcdB(F) in denaturant solutions are reported in detail, the corresponding information on the chromosomal CcdB(Vfi) is rather scarce. Therefore, we studied urea-induced unfolding of CcdB(Vfi) at various temperatures and protein concentrations by circular dichroism spectroscopy. Global model analysis of spectroscopic data suggests that CcdB(Vfi) dimer unfolds to the corresponding monomeric components in a reversible two-state manner. Results reveal that at physiological temperatures CcdB(Vfi) exhibits lower thermodynamic stability compared to CcdB(F). At high urea concentrations CcdB(Vfi), similarly to CcdB(F), retains a significant amount of secondary structure. Differences in thermodynamic parameters of CcdB(Vfi) and CcdB(F) unfolding can reasonably be explained by the differences in their structural features

What drives the binding of minor groove-directed ligands to DNA hairpins?

Understanding the molecular basis of ligand–DNA-binding events, and its application to the rational design of novel drugs, requires knowledge of the structural features and forces that drive the corresponding recognition processes. Existing structural evidence on DNA complexation with classical minor groove-directed ligands and the corresponding studies of binding energetics have suggested that this type of binding can be described as a rigid-body association. In contrast, we show here that the binding-coupled conformational changes may be crucial for the interpretation of DNA (hairpin) association with a classical minor groove binder (netropsin). We found that, although the hairpin form is the only accessible state of ligand-free DNA, its association with the ligand may lead to its transition into a duplex conformation. It appears that formation of the fully ligated duplex from the ligand-free hairpin, occurring via two pathways, is enthalpically driven and accompanied by a significant contribution of the hydrophobic effect. Our thermodynamic and structure-based analysis, together with corresponding theoretical studies, shows that none of the predicted binding steps can be considered as a rigid-body association. In this light we anticipate our thermodynamic approach to be the basis of more sophisticated nucleic acid recognition mechanisms, which take into account the dynamic nature of both the nucleic acid and the ligand molecule

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Additional file 14. A table with description of strains and plasmids generated and used in this study and graphical representation of over-expressed operons/genes

Designed folding pathway of modular coiled-coil-based proteins

Natural proteins are characterised by a complex folding pathway defined uniquely for each fold. Designed coiled-coil protein origami (CCPO) cages are distinct from natural compact proteins, since their fold is prescribed by discrete long-range interactions between orthogonal pairwise-interacting coiled-coil (CC) modules within a single polypeptide chain. Here, we demonstrate that CCPO proteins fold in a stepwise sequential pathway. Molecular dynamics simulations and stopped-flow Förster resonance energy transfer (FRET) measurements reveal that CCPO folding is dominated by the effective intra-chain distance between CC modules in the primary sequence and subsequent folding intermediates, allowing identical CC modules to be employed for multiple cage edges and thus relaxing CCPO cage design requirements. The number of orthogonal modules required for constructing a CCPO tetrahedron can be reduced from six to as little as three different CC modules. The stepwise modular nature of the folding pathway offers insights into the folding of tandem repeat proteins and can be exploited for the design of modular protein structures based on a given set of orthogonal modules

Modulation of Coiled-Coil Dimer Stability through Surface Residues while Preserving Pairing Specificity

The coiled-coil dimer is a widespread protein structural motif and, due to its designability, represents an attractive building block for assembling modular nanostructures. The specificity of coiled-coil dimer pairing is mainly based on hydrophobic and electrostatic interactions between residues at positions a, d, e, and g of the heptad repeat. Binding affinity, on the other hand, can also be affected by surface residues that face away from the dimerization interface. Here we show how design of the local helical propensity of interacting peptides can be used to tune the stabilities of coiled-coil dimers over a wide range. By designing intramolecular charge pairs, regions of high local helical propensity can be engineered to form trigger sequences, and dimer stability is adjusted without changing the peptide length or any of the directly interacting residues. This general principle is demonstrated by a change in thermal stability by more than 30 °C as a result of only two mutations outside the binding interface. The same approach was successfully used to modulate the stabilities in an orthogonal set of coiled-coils without affecting their binding preferences. The stability effects of local helical propensity and peptide charge are well described by a simple linear model, which should help improve current coiled-coil stability prediction algorithms. Our findings enable tuning the stabilities of coiled-coil-based building modules match a diverse range of applications in synthetic biology and nanomaterials

Energetic Basis of Uncoupling Folding from Binding for an Intrinsically Disordered Protein

Intrinsically disordered proteins (IDPs) are proteins that lack a unique three-dimensional structure in their native state. Many have, however, been found to fold into a defined structure when interacting with specific binding partners. The energetic implications of such behavior have been widely discussed, yet experimental thermodynamic data is scarce. We present here a thorough thermodynamic and structural study of the binding of an IDP (antitoxin CcdA) to its molecular target (gyrase poison CcdB). We show that the binding-coupled folding of CcdA is driven by a combination of specific intramolecular interactions that favor the final folded structure and a less specific set of intermolecular contacts that provide a desolvation entropy boost. The folded structure of the bound IDP appears to be defined largely by its own amino acid sequence, with the binding partner functioning more as a facilitator than a mold to conform to. On the other hand, specific intermolecular interactions do increase the binding affinity up to the picomolar range. Overall, this study shows how an IDP can achieve very strong and structurally well-defined binding and it provides significant insight into the molecular forces that enable such binding properties