Structural and functional characterization of the pore-forming domain of pinholin S2168.

Pinholin S2168 triggers the lytic cycle of bacteriophage φ21 in infected Escherichia coli Activated transmembrane dimers oligomerize into small holes and uncouple the proton gradient. Transmembrane domain 1 (TMD1) regulates this activity, while TMD2 is postulated to form the actual "pinholes." Focusing on the TMD2 fragment, we used synchrotron radiation-based circular dichroism to confirm its α-helical conformation and transmembrane alignment. Solid-state 15N-NMR in oriented DMPC bilayers yielded a helix tilt angle of τ = 14°, a high order parameter (Smol = 0.9), and revealed the azimuthal angle. The resulting rotational orientation places an extended glycine zipper motif (G40xxxS44xxxG48) together with a patch of H-bonding residues (T51, T54, N55) sideways along TMD2, available for helix-helix interactions. Using fluorescence vesicle leakage assays, we demonstrate that TMD2 forms stable holes with an estimated diameter of 2 nm, as long as the glycine zipper motif remains intact. Based on our experimental data, we suggest structural models for the oligomeric pinhole (right-handed heptameric TMD2 bundle), for the active dimer (right-handed Gly-zipped TMD2/TMD2 dimer), and for the full-length pinholin protein before being triggered (Gly-zipped TMD2/TMD1-TMD1/TMD2 dimer in a line).

A Phosphine Functionalized ß-Diketimine Ligand for the Synthesis of Manifold Metal Complexes.

A bis(diphenyl)-phosphine functionalized ß-diketimine (PNac-H) was synthesized as a flexible ligand for transition metal complexes. The newly designed ligand features symmetrically placed phosphine moieties around a ß-diketimine unit, forming a PNNP-type pocket. Due to the hard and soft donor atoms (N vs. P) the ligand can stabilize various coordination polyhedra. A complete series ranging from coordination numbers 2 to 6 was realized. Linear, trigonal planar, square planar, tetrahedral, square pyramidal, and octahedral coordination arrangements containing the PNac-ligand around the metal center were observed by using suitable metal sources. Hereby, PNac-H or its anion PNac- acts as mono-, bi- and tetradendate ligand. Such a broad flexibility is unusual for a rigid tetradentate system. The structural motifs were realized by treatment of PNac-H with a series of late transition metal precursors, for example, silver, gold, nickel, copper, platinum, and rhodium. The new complexes have been fully characterized by single crystal X-ray diffraction, NMR, IR, UV/Vis spectroscopy, mass spectrometry as well as elemental analysis. Additionally, selected complexes were investigated regarding their photophysical properties. Thus, PNac-H proved to be an ideal ligand platform for the selective coordination and stabilization of various metal ions in diverse polyhedra and oxidation states.

Development of an Aptamer-Based QCM-D Biosensor for the Detection of Thrombin Using Supported Lipid Bilayers as Surface Functionalization.

Biosensors play an important role in numerous research fields. Quartz crystal microbalances with dissipation monitoring (QCM-Ds) are sensitive devices, and binding events can be observed in real-time. In combination with aptamers, they have great potential for selective and label-free detection of various targets. In this study, an alternative surface functionalization for a QCM-D-based aptasensor was developed, which mimics an artificial cell membrane and thus creates a physiologically close environment for the binding of the target to the sensor. Vesicle spreading was used to form a supported lipid bilayer (SLB) of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphethanolamine-N-(cap biotinyl) (biotin-PE). The SLB was then coated with streptavidin followed by applying a biotinylated aptamer against thrombin. SLB formation was investigated in terms of temperature and composition. Temperatures of 25 °C and below led to incomplete SLB formation, whereas a full bilayer was built at higher temperatures. We observed only a small influence of the content of biotinylated lipids in the mixture on the further binding of streptavidin. The functionalization of the sensor surface with the thrombin aptamer and the subsequent thrombin binding were investigated at different concentrations. The sensor could be reconstituted by incubation with a 5 M urea solution, which resulted in the release of the thrombin from the sensor surface. Thereafter, it was possible to rebind thrombin. Thrombin in spiked samples of human serum was successfully detected. The developed system can be easily applied to other target analytes using the desired aptamers.