Faculty Bibliography

A wide range of light-activated molecules (photoswitches and phototriggers) have been used to the study of computational properties of an isolated neuron by acting pre and postsynaptically. However, new tools are being pursued to elicit a presynaptic calcium influx that triggers the release of neurotransmitters, most of them based in calcium-permeable Channelrhodopsin-2 mutants. Here we describe a method to control exocytosis of synaptic vesicles through the use of a light-gated glutamate receptor (LiGluR), which has recently been demonstrated that supports secretion by means of calcium influx in chromaffin cells. Expression of LiGluR in hippocampal neurons enables reversible control of neurotransmission with light, and allows modulating the firing rate of the postsynaptic neuron with the wavelength of illumination. This method may be useful for the determination of the complex transfer function of individual synapses.

Pericyclic reaction cascades are unparalleled in their ability to quickly generate complex structures with excellent stereocontrol. Herein, the use of a biomimetic Stille/8pi electrocyclization/Diels-Alder cascade to successfully assemble the core structure of (-)-PF-1018 is reported.

A short and asymmetric entry to the core structure of the cordiachromes has been developed, allowing access to (-)-isoglaziovianol in seven steps. Our synthesis includes a Trost asymmetric allylic alkylation and a reaction cascade triggered by a vinyl quinone Diels-Alder reaction and followed by intramolecular nucleophilic interception.

The use of azobenzene photoswitches has become a dependable method for rapid and exact modulation of biological processes and material science systems. The requirement of ultraviolet light for azobenzene isomerization is not ideal for biological systems due to poor tissue penetration and potentially damaging effects. While modified azobenzene cores with a red-shifted cis-to-trans isomerization have been previously described, they have not yet been incorporated into a powerful method to control protein function: the photoswitchable tethered ligand (PTL) approach. We report the synthesis and characterization of a red-shifted PTL, L-MAG0460, for the light-gated ionotropic glutamate receptor LiGluR. In cultured mammalian cells, the LiGluR+L-MAG0460 system is activated rapidly by illumination with 400-520 nm light to generate a large ionic current. The current rapidly turns off in the dark as the PTL relaxes thermally back to the trans configuration. The visible light excitation and single-wavelength behavior considerably simplify use and should improve utilization in tissue.

The undecose nucleoside antibiotics herbicidin C and aureonuclemycin are biologically highly active and represent challenging targets for total synthesis. Herein, the gradual evolution of our synthetic strategy toward these natural products is described in detail. The initial route encompasses metalate addition chemistry but suffers from poor stereochemical control. In contrast, the ultimately successful strategy benefits from a variety of reagent-controlled stereoselective transformations, including a surprisingly facile and highly diastereoselective N-glycosylation process. The presented work also describes new building blocks that might find further application in carbohydrate chemistry.

Tetraphenylporphyrin derivatives represent a promising class of high-affinity ligands for voltage-gated potassium (Kv) channels. Herein, we investigated the mode of Kv channel block of one tetraphenylporphyrin derivative, por3, using electrophysiological methods, structure-based mutagenesis, and solid-state NMR spectroscopy. The combined data showed that por3 specifically blocks Kv1.x channels. Unexpectedly, 2 different por3 binding modes lead to Kv1.x channel block exerted through multiple por3 binding sites: first, por3 interacts in a highly cooperative and specific manner with the voltage sensor domain stabilizing closed Kv1 channel state(s). Therefore, stronger depolarization is needed to activate Kv1.x channels in the presence of por3. Second, por3 bind to a single site at the external pore entrance to block the ion conduction pathway of activated Kv1.x channels. This block is voltage-independent. Por3 appears to have equal affinities for voltage-sensor and pore. However, at negative voltage and low por3 concentration, por3 gating modifier properties prevail due to the high cooperativity of binding. By contrast, at positive voltages, when Kv1.x channels are fully activated, por3 pore blocking properties predominate.

UNLABELLED: Cell-cell signaling in Xylella fastidiosa has been implicated in the coordination of traits enabling colonization in plant hosts as well as insect vectors. This cell density-dependent signaling has been attributed to a diffusible signaling factor (DSF) produced by the DSF synthase RpfF. DSF produced by related bacterial species are unsaturated fatty acids, but that of X. fastidiosa was thought to be different from those of other taxa. We describe here the isolation and characterization of an X. fastidiosa DSF (XfDSF) as 2(Z)-tetradecenoic acid. This compound was isolated both from recombinant Erwinia herbicola expressing X. fastidiosa rpfF and from an X. fastidiosa rpfC deletion mutant that overproduces DSF. Since an rpfF mutant is impaired in biofilm formation and underexpresses the hemagglutinin-like protein-encoding genes hxfA and hxfB, we demonstrate that these traits can be restored by ca. 0.5 microM XfDSF but not by myristic acid, the fully saturated tetradecenoic acid. A phoA-based X. fastidiosa biosensor that assesses DSF-dependent expression of hxfA or hxfB revealed a high level of molecular specificity of DSF signaling. IMPORTANCE: X. fastidiosa causes diseases in many important plants, including grape, where it incites Pierce's disease. Virulence of X. fastidiosa for grape is coordinated by cell-cell signaling molecules, designated DSF (Diffusible Signaling Factor). Mutants blocked in DSF production are hypervirulent for grape, suggesting that virulence is suppressed upon DSF accumulation and that disease could be controlled by artificial elevation of the DSF level in plants. In this work, we describe the isolation of the DSF produced by X. fastidiosa and the verification of its biological activity as an antivirulence factor. We also have developed X. fastidiosa DSF biosensors to evaluate the specificity of cell-cell signaling to be investigated.