Quantitative analysis of the effect of derivatisation of [Ru(BPY) phen] with a quinoline moiety on the interaction with DNA

The bifunctional Ru(II) complex [Ru(BPY)2POQ-Nmet]2+ (1), in which the metallic unit is tethered by an aliphatic chain to an organic DNA binder, was designed in order to increase the affinity toward nucleic acids. The interaction of 1 with DNA was characterised from luminescence and absorption data and compared with the binding of its monofunctional metallic and organic analogues, [Ru(BPY)2(ac)phen]2+ (2) and Nmet-quinoline (3). The bifunctional complex has a binding affinity one order of magnitude higher than that of each of its separated moieties. Absorption changes induced upon addition of DNA at different pH indicate protonation of the organic sub-unit upon interaction with DNA under neutral conditions. The combination of the luminescence data under steady-state and time-resolved conditions shows that the attachment of the organic unit in 1 induces modifications of the association modes of the metallic unit, owing to the presence of the aliphatic chain which probably hinders the metallic moiety binding. The salt dependence of the binding constants was analysed in order to compare the thermodynamic parameters describing the association with DNA for each complex. This study demonstrates the interest of the derivatisation of a Ru(II) complex with an organic moiety (ia the bifunctional ligand POQ-Nmet) for the development of high affinity DNA probes or photoreactive agents.

The F4 fimbrial chaperone FaeE is stable as a monomer that does not require self-capping of its pilin-interactive surfaces.

Many Gram-negative bacteria use the chaperone-usher pathway to express adhesive surface structures, such as fimbriae, in order to mediate attachment to host cells. Periplasmic chaperones are required to shuttle fimbrial subunits or pilins through the periplasmic space in an assembly-competent form. The chaperones cap the hydrophobic surface of the pilins through a donor-strand complementation mechanism. FaeE is the periplasmic chaperone required for the assembly of the F4 fimbriae of enterotoxigenic Escherichia coli. The FaeE crystal structure shows a dimer formed by interaction between the pilin-binding interfaces of the two monomers. Dimerization and tetramerization have been observed previously in crystal structures of fimbrial chaperones and have been suggested to serve as a self-capping mechanism that protects the pilin-interactive surfaces in solution in the absence of the pilins. However, thermodynamic and biochemical data show that FaeE occurs as a stable monomer in solution. Other lines of evidence indicate that self-capping of the pilin-interactive interfaces is not a mechanism that is conservedly applied by all periplasmic chaperones, but is rather a case-specific solution to cap aggregation-prone surfaces.

Solution structure of the N-terminal transactivation domain of ERM modified by SUMO-1.

ERM is a member of the PEA3 group of the Ets transcription factor family that plays important roles in development and tumorigenesis. The PEA3s share an N-terminal transactivation domain (TADn) whose activity is inhibited by small ubiquitin-like modifier (SUMO). However, the consequences of sumoylation and its underlying molecular mechanism remain unclear. The domain structure of ERM TADn alone or modified by SUMO-1 was analyzed using small-angle X-ray scattering (SAXS). Low resolution shapes determined ab initio from the scattering data indicated an elongated shape and an unstructured conformation of TADn in solution. Covalent attachment of SUMO-1 does not perturb the structure of TADn as indicated by the linear arrangement of the SUMO moiety with respect to TADn. Thus, ERM belongs to the growing family of proteins that contain intrinsically unstructured regions. The flexible nature of TADn may be instrumental for ERM recognition and binding to diverse molecular partners.

Expression, purification and X-ray crystallographic analysis of the Helicobacter pylori blood group antigen-binding adhesin BabA

Helicobacter pylori is a human pathogen that colonizes about 50% of the world's population, causing chronic gastritis, duodenal ulcers and even gastric cancer. A steady emergence of multiple antibiotic resistant strains poses an important public health threat and there is an urgent requirement for alternative therapeutics. The blood group antigen-binding adhesin BabA mediates the intimate attachment to the host mucosa and forms a major candidate for novel vaccine and drug development. Here, the recombinant expression and crystallization of a soluble BabA truncation (BabA^25-460) corresponding to the predicted extracellular adhesin domain of the protein are reported. X-ray diffraction data for nanobody-stabilized BabA^25-460 were collected to 2.25Å resolution from a crystal that belonged to space group P21, with unit-cell parameters a = 50.96, b = 131.41, c = 123.40Å, α = 90.0, β = 94.8, γ = 90.0°, and which was predicted to contain two BabA^25-460-nanobody complexes per asymmetric unit.