2016, Lee, Yan-Jiun, Schmidt, Moritz J., Tharp, Jeffery M., Weber, Annemarie, Koenig, Amber L., Zheng, Hong, Gao, Jianmin, Waters, Marcey L., Summerer, Daniel, Liu, Wenshe R.

Fluorophenylalanines bearing 2-5 fluorine atoms at the phenyl ring have been genetically encoded by amber codon. Replacement of F59, a phenylalanine residue that is directly involved in interactions with trimethylated K9 of histone H3, in the Mpp8 chromodomain recombinantly with fluorophenylalanines significantly impairs the binding to a K9-trimethylated H3 peptide.

2013, Schildknecht, Stefan, Weber, Annemarie, Gerding, Hanne R., Pape, Regina, Robotta, Marta, Drescher, Malte, Marquardt, Andreas, Daiber, Andreas, Ferger, Boris, Leist, Marcel

NADPH oxidases (NOX), catalyzing the reduction of molecular oxygen to form the superoxide radical anion (^•O₂ ^-) and hydrogen peroxide (H₂O₂), are involved in several pathological conditions, such as stroke, diabetes, atherosclerosis, but also in chronic neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, or multiple sclerosis. GKT136901 is a novel NOX-1/4 inhibitor with potential application in the areas of diabetic nephropathy, stroke, or neurodegeneration. In the present study, we investigated additional pharmacological activities of the compound with respect to direct free radical scavenging. GKT136901 did not interact with nitric oxide (^•NO), ^•O₂ ^-, or hydroxyl radicals (^•OH), but it acted as selective scavenger of peroxynitrite (PON) already in the submicromolar concentration range. Alpha synuclein (ASYN) is a protein involved in the pathogenesis of Parkinson's disease and a known target for PON-dependent tyrosine nitration. Submicromolar concentrations of GKT136901 prevented tyrosine nitration and di-tyrosine-dependent dimer formation of ASYN by PON as indicated by Western blot and mass spectrometric analysis. GKT136901 itself was degraded when exposed to PON. In a human neuronal cell model, GKT136901 prevented both the depletion of reduced intracellular glutathione, and the degeneration of neurites when present during PON treatment of the cells. When GKT136901 was applied after PON treatment, no protective effect was observed, thus excluding an impact of GKT136901 on cellular death/survival pathways. In summary, selective scavenging of PON is an additional pharmacological property of the NOX-1/4 inhibitor GKT136901, and this may add to the efficiency of the drug in several disease models.

2014, Schmidt, Moritz J., Weber, Annemarie, Pott, Moritz, Welte, Wolfram, Summerer, Daniel

The site-selective introduction of photo-crosslinking groups into proteins enables the discovery and mapping of weak and/or transient protein interactions with high spatiotemporal resolution, both in vitro and in vivo. We report the genetic encoding of a furan-based, photo-crosslinking amino acid in human cells; it can be activated with red light, thus offering high penetration depths in biological samples. This is achieved by activation of the amino acid and charging to its cognate tRNA by a pyrrolysyl-tRNA-synthetase (PylRS) mutant with broad polyspecificity. To gain insights into the recognition of this amino acid and to provide a rationale for its polyspecificity, we solved three crystal structures of the PylRS mutant: in its apo-form, in complex with adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP) and in complex with the AMP ester of the furan amino acid. These structures provide clues for the observed polyspecificity and represent a promising starting point for the engineering of PylRS mutants with further increased substrate scope.

2010, Domingo Köhler, Silvia, Weber, Annemarie, Howard, S. Peter, Welte, Wolfram, Drescher, Malte

TonB from Escherichia coli and its homologues are critical for the uptake of siderophores through the outer membrane of Gram-negative bacteria using chemiosmotic energy. When different models for the mechanism of TonB mediated energy transfer from the inner to the outer membrane are discussed, one of the key questions is whether TonB spans the periplasm. In this article, we use long range distance measurements by spin-label pulsed EPR (Double Electron Electron Resonance, DEER) and CD spectroscopy to show that the proline-rich segment of TonB exists in a PPII-like conformation. The result implies that the proline-rich segment of TonB possesses a length of more than 15 nm, sufficient to span the periplasm of Gram-negative bacteria.

2013-08-20, Damnjanovic, Bojana, Weber, Annemarie, Odermatt, Meike, Greie, Jörg-Christian, Apell, Hans-Jürgen

The high-affinity potassium uptake system KdpFABC is a unique type Ia P-type ATPase, because it separates the sites of ATP hydrolysis and ion transport on two different subunits. KdpFABC was expressed in Escherichia coli. It was then isolated and purified to homogeneity to obtain a detergent-solubilized enzyme complex that allowed the analysis of ion binding properties. The electrogenicity and binding affinities of the ion pump for K+ and H+ were determined in detergent-solubilized complexes by means of the electrochromic styryl dye RH421. Half-saturating K+ concentrations and pK values for H+ binding could be obtained in both the unphosphorylated and phosphorylated conformations of KdpFABC. The interaction of both ions with KdpFABC was studied in detail, and the presence of independent binding sites was ascertained. It is proposed that KdpFABC reconstituted in vesicles translocates protons at a low efficiency opposite from the well-established import of K+ into the bacteria. On the basis of our results, various mechanistic pump cycle models were derived from the general Post–Albers scheme of P-type ATPases and discussed in the framework of the experimental evidence to propose a possible molecular pump cycle for KdpFABC.