Chemical Proteomic Profiling of Protein 4′‐Phosphopantetheinylation in Mammalian Cells

Protein 4′‐phosphopantetheinylation is an essential post‐translational modification (PTM) in prokaryotes and eukaryotes. So far, only five protein substrates of this specific PTM have been discovered in mammalian cells. These proteins are known to perform important functions, including fatty acid biosynthesis and folate metabolism, as well as β‐alanine activation. To explore existing and new substrates of 4′‐phosphopantetheinylation in mammalian proteomes, we designed and synthesized a series of new pantetheine analogue probes, enabling effective metabolic labelling of 4′‐phosphopantetheinylated proteins in HepG2 cells. In combination with a quantitative chemical proteomic platform, we enriched and identified all the currently known 4′‐phosphopantetheinylated proteins with high confidence, and unambiguously determined their exact sites of modification. More encouragingly, we discovered, using targeted chemical proteomics, a potential 4′‐phosphopantetheinylation site in the protein of mitochondrial dehydrogenase/reductase SDR family member 2 (DHRS2).     

Double Histidine Based EPR Measurements at Physiological Temperatures Permit Site-Specific Elucidation of Hidden Dynamics in Enzymes

Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis-Cu^IINTA label to obtain site-specific information on dynamics for both an α-helix and β-sheet site of GB1, the immunoglobulin binding domain of protein G. Spectral features found in our CW-EPR measurements were consistent with the overall rigid nature of GB1 and with predictions from molecular dynamics simulations. Using this information, we show the potential of this approach to elucidate the role of dynamics in substrate binding of a functionally necessary α-helix in human glutathione transferase A1-1 (hGSTA1-1). We observe two dynamical modes for the helix. The addition of the inhibitor GS-Met and GS-Hex resulted in hGSTA1-1 to favor the more rigid active state conformation, while the faster mode potentially aids the search for substrates. Together the results illustrate the remarkable potential of the dHis-based labelling approach to measure site-specific dynamics using room temperature lineshape analysis.     

Versatile Bispidine-Based Bifunctional Chelators for 64CuII-Labelling of Biomolecules

Bifunctional chelators as parts of modular metal-based radiopharmaceuticals are responsible for stable complexation of the radiometal ion and for covalent linkage between the complex and the targeting vector. To avoid loss of complex stability, the bioconjugation strategy should not interfere with the radiometal chelation by occupying coordinating groups. The C9 position of the very stable Cu^II chelator 3,7-diazabicyclo[3.3.1]nonane (bispidine) is virtually predestined to introduce functional groups for facile bioconjugation as this functionalisation does not disturb the metal binding centre. We describe the preparation and characterisation of a set of novel bispidine derivatives equipped with suitable functional groups for diverse bioconjugation reactions, including common amine coupling strategies (bispidine-isothiocyanate) and the Cu-free strain-promoted alkyne–azide cycloaddition. We demonstrate their functionality and versatility in an exemplary way by conjugation to an antibody-based biomolecule and validate the obtained conjugate in vitro and in vivo.     

Tuning of Oxidation Potential of Ferrocene for Ratiometric Redox Labeling and Coding of Nucleotides and DNA

Three sets of 7-deazaadenine and cytosine nucleosides and nucleoside triphosphates bearing either unsubstituted ferrocene, octamethylferrocene and ferrocenecarboxamide linked through an alkyne tether to position 7 or 5, respectively, were designed and synthesized. The modified dN^FcXTP s were good substrates for KOD XL DNA polymerase in primer extension and were used for enzymatic synthesis of redox-labelled DNA probes. Square-wave voltammetry showed that the octamethylferrocene oxidation potential was shifted to lower values, whilst the ferrocenecarboxamide was shifted to higher potentials, as compared to ferrocene. Tailed PEX products containing different ratios of Fc-labelled A ( dA^Fc ) and FcPa-labelled C ( dC^FcPa ) were synthesized and hybridized with capture oligonucleotides immobilized on gold electrodes to study the electrochemistry of the redox-labelled DNA. Clearly distinguishable, fully orthogonal and ratiometric peaks were observed for the dA^Fc and dC^FcPa bases in DNA, demonstrating their potential for use in redox coding of nucleobases and for the direct electrochemical measurement of the relative ratio of nucleobases in an unknown sequence of DNA.     

关于p3n的优美性

设G(V,E)是一个简单图,对自然数k,当V(Gk)=V(G),E(Gk)=E(G)∪{uv|d(u,v)=k},则称图Gk为k-次方图.本文证明了图P3n的优美性.     

Zur Reindarstellung von tritiummarkierten Pyrimidinderivaten 3. Mitteilung: Darstellung von 5-Brom-[6-3H]uracil und [6-3H]Uracil

Das Phanomen der Lyolumineszenz wurde erstmuls 1895 von Wiedemann und Schmidt an Alkalihalogeniden nach Bestrahlung mit Katodenstrahlcn beobachtet. In ihrer Arbeit “Uber Luminescenz", die in den ”Annalen der Physik und Chemie" erschien, schreiben sie [24]:

“Ferner haben wir eine weitero Art der Luminescenz aufgefunden, welche man mit dem Namen Lyoluininescenz belegen konnto. In gewissen Fallen tritt beim Losen von Substanzen eine Lichtentwicklung auf, nnchdem sie vorher bestrablt worden sind, wahrend sie eine solche ohne Bestrahlung nicht zeigen“.     

Soil matrix tracer contamination and canopy recycling did not impair 13CO2 plant–soil pulse labelling experiments

When conducting ¹³CO₂ plant–soil pulse labelling experiments, tracer material might cause unwanted side effects which potentially affect δ¹³C measurements of soil respiration (δ¹³C_SR) and the subsequent data interpretation. First, when the soil matrix is not isolated from the atmosphere, contamination of the soil matrix with tracer material occurs leading to a physical back-diffusion from soil pores. Second, when using canopy chambers continuously, ¹³CO₂ is permanently re-introduced into the atmosphere due to leaf respiration which then aids re-assimilation of tracer material by the canopy. Accordingly, two climate chamber experiments on European beech saplings (Fagus sylvatica L.) were conducted to evaluate the influence of soil matrix ¹³CO₂ contamination and canopy recycling on soil ¹³CO₂ efflux during ¹³CO₂ plant–soil pulse labelling experiments. For this purpose, a combined soil/canopy chamber system was developed which separates soil and canopy compartments in order to (a) prevent diffusion of ¹³C tracer into the soil chamber during a ¹³CO₂ canopy pulse labelling and (b) study stable isotope processes in soil and canopy individually and independently. In combination with laser spectrometry measuring CO₂ isotopologue mixing ratios at a rate of 1 Hz, we were able to measure δ¹³C in canopy and soil at very high temporal resolution. For the soil matrix contamination experiment, ¹³CO₂ was applied to bare soil, canopy only or, simultaneously, to soil and canopy of the beech trees. The obtained δ¹³C_SR fluxes from the different treatments were then compared with respect to label re-appearance, first peak time and magnitude. By determining the δ¹³C_SR decay of physical ¹³CO₂ back-diffusion from bare soils (contamination), it was possible to separate biological and physical components in δ¹³C_SR of a combined flux of both. A second pulse labelling experiment, with chambers permanently enclosing the canopy, revealed that ¹³CO₂ recycling at canopy level had no effect on δ¹³C_SR dynamics.     

Estimation of groundwater recharge via deuterium labelling in the semi-arid Cuvelai-Etosha Basin,Namibia

The stable water isotope deuterium (²H) was applied as an artificial tracer (²H₂O) in order to estimate groundwater recharge through the unsaturated zone and describe soil water movement in a semi-arid region of northern central Namibia. A particular focus of this study was to assess the spatiotemporal persistence of the tracer when applied in the field on a small scale under extreme climatic conditions and to propose a method to obtain estimates of recharge in data-scarce regions. At two natural sites that differ in vegetation cover, soil and geology, 500?ml of a 70?% ²H₂O solution was irrigated onto water saturated plots. The displacement of the ²H peak was analyzed 1 and 10 days after an artificial rain event of 20 mm as well as after the rainy season. Results show that it is possible to apply the peak displacement method for the estimation of groundwater recharge rates in semi-arid environments via deuterium labelling. Potential recharge for the rainy season 2013/2014 was calculated as 45 mm a^?1 at 5.6 m depth and 40 mm a^?1 at 0.9 m depth at the two studied sites, respectively. Under saturated conditions, the artificial rain events moved 2.1 and 0.5 m downwards, respectively. The tracer at the deep sand site (site 1) was found after the rainy season at 5.6 m depth, corresponding to a displacement of 3.2 m. This equals in an average travel velocity of 2.8 cm d^?1 during the rainy season at the first site. At the second location, the tracer peak was discovered at 0.9 m depth; displacement was found to be only 0.4 m equalling an average movement of 0.2 cm d^?1 through the unsaturated zone due to an underlying calcrete formation. Tracer recovery after one rainy season was found to be as low as 3.6?% at site 1 and 1.9?% at site 2. With an in situ measuring technique, a three-dimensional distribution of ²H after the rainy season could be measured and visualized. This study comprises the first application of the peak displacement method using a deuterium labelling technique for the estimation of groundwater recharge in semi-arid regions. Deuterium proved to be a suitable tracer for studies within the soil–vegetation–atmosphere interface. The results of this study are relevant for the design of labelling experiments in the unsaturated zone of dry areas using ²H₂O as a tracer and obtaining estimations of groundwater recharge on a local scale. The presented methodology is particularly beneficial in data-scarce environments, where recharge pathways and mechanisms are poorly understood.     

Infrared Study of Anthraquinone-Anion-Radical and its 180 Labelled Isomer

Abstract

Infrared spectra of anthraquinone and anthraquinone-¹⁸0-anion-radicals have been investigated. Anion-radicals have been obtained by electrochemical reduction of anthraquinone and anthraquinone-¹⁸0 in dimethylsulf-oxide-d₆.

Band assignment has been performed using the group vibrational concept and isotopic shift data. The assignment of IR active C[dbnd]O stretching band and the other bands in 1150–1800 cm^?1 region of anthraquinone anion-radical (anthrasemiquinone) have been discussed and compared with those of some other ¹⁸0 labelled anion-radicals. A decrease of 182 cm^?1 has been found forafter transformation of the neutral quinone into anion-radical.