Atomic-level structure characterization of an ultrafast folding mini-protein denatured state

Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing 15N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R2 rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding

High-resolution NMR structure of an RNA model system : the 14-mer cUUCGg tetraloop hairpin RNA

We present a high-resolution nuclear magnetic resonance (NMR) solution structure of a 14-mer RNA hairpin capped by cUUCGg tetraloop. This short and very stable RNA presents an important model system for the study of RNA structure and dynamics using NMR spectroscopy, molecular dynamics (MD) simulations and RNA force-field development. The extraordinary high precision of the structure (root mean square deviation of 0.3 Å) could be achieved by measuring and incorporating all currently accessible NMR parameters, including distances derived from nuclear Overhauser effect (NOE) intensities, torsion-angle dependent homonuclear and heteronuclear scalar coupling constants, projection-angle-dependent cross-correlated relaxation rates and residual dipolar couplings. The structure calculations were performed with the program CNS using the ARIA setup and protocols. The structure quality was further improved by a final refinement in explicit water using OPLS force field parameters for non-bonded interactions and charges. In addition, the 2'-hydroxyl groups have been assigned and their conformation has been analyzed based on NOE contacts. The structure currently defines a benchmark for the precision and accuracy amenable to RNA structure determination by NMR spectroscopy. Here, we discuss the impact of various NMR restraints on structure quality and discuss in detail the dynamics of this system as previously determined

Der Deutsche Germanistenverband –: eine „Fachgenossenschaft“ im Zielkonflikt

Deutscher Germanistenverband: An Academic Association in a Conflict of Objectives From its inauguration in 1912 until 1933 the DGV resembled an ideological society successfully pursuing anti-modern nationalistic goals in the area of educational and school policy with modern methods. At best, it might be said to have only marginally fulfilled the role of an academic association for the field of study. While it did not actively seek to join the National Socialist movement, it did not resist it either. Only since its annually held conference in966 has the DGV started to deal with its past and subsequently reformed the association’s activities rigorously to become a modern academic association. What remains problematic today, is the fact that the DGV represents both the interests of German scholars from higher education and those of school teachers of German – which in fact frequently diverge from each other – and therefore, due to internal differentiation, is in danger of being reduced to being an umbrella association

Annual Report 2010 - Institute of Radiochemistry

At the beginning of 2011, the former Forschungszentrum Dresden-Rossendorf (FZD) was fully integrated into the Helmholtz Association, as Helmholtz-Zentrum Dresden-Rossendorf (HZDR). Therefore, the present Annual Report 2010 of the Institute of Radiochemistry (IRC) is published as the first HZDR-Report. The Institute of Radiochemistry is one of the six Research Institutes of this centre. IRC contributes to the research program “Nuclear Safety Research” in the “Research Field of Energy” and performs basic and applied research in radiochemistry and radioecology. Motivation and background of our research are environmental processes relevant for the installation of nuclear waste repositories, for remediation of uranium mining and milling sites, and for radioactive contaminations caused by nuclear accidents and fallout. Because of their high radiotoxicity and long half-life the actinides are of special interest

Alkylated albumin-derived dipeptide C(-HETE)P derivatized by propionic anhydride as a biomarker for the verification of poisoning with sulfur mustard

Sulfur mustard (SM) is a banned chemical warfare agent recently used in the Syrian Arab Republic conflict causing erythema and blisters characterized by complicated and delayed wound healing. For medical and legal reasons, the proof of exposure to SM is of high toxicological and forensic relevance. SM reacts with endogenous human serum albumin (HSA adducts) alkylating the thiol group of the cysteine residue C34, thus causing the addition of the hydroxyethylthioethyl (HETE) moiety. Following proteolysis with pronase, the biomarker dipeptide C(-HETE)P is produced. To expand the possibilities for verification of exposure, we herein introduce a novel biomarker produced from that alkylated dipeptide by derivatization with propionic anhydride inducing the selective propionylation of the N-terminus yielding PA-C(-HETE)P. Quantitative derivatization is carried out at room temperature in aqueous buffer within 10 s. The biomarker was found to be stable in the autosampler at 15 °C for at least 24 h, thus documenting its suitability even for larger sets of samples. Selective and sensitive detection is done by micro liquid chromatography-electrospray ionization tandem-mass spectrometry (μLC-ESI MS/MS) operating in the selected reaction monitoring (SRM) mode detecting product ions of the single protonated PA-C(-HETE)P (m/z 379.1) at m/z 116.1, m/z 137.0, and m/z 105.0. The lower limit of detection corresponds to 32 nM SM in plasma in vitro and the limit of identification to 160 nM. The applicability to real exposure scenarios was proven by analyzing samples from the Middle East confirming poisoning with SM. Graphical abstractDeutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Peer Reviewe

Near-infrared absorption properties of oxygen-rich stardust analogues: The influence of coloring metal ions

Several astrophysically relevant solid oxides and silicates have extremely small opacities in the visual and near-infrared in their pure forms. Datasets for the opacities and for the imaginary part k of their complex indices of refraction are hardly available in these wavelength ranges. We aimed at determining k for spinel, rutile, anatase, and olivine, especially in the near-infrared region. Our measurements were made with impurity-containing, natural, and synthetic stardust analogs. Two experimental methods were used: preparing small sections of natural minerals and synthesizing melt droplets under the electric arc furnace. In both cases, the aborption properties of the samples were measured by transmission spectroscopy. For spinel (MgAl2O4), anatase, rutile (both TiO2), and olivine ((Mg,Fe)2SiO4), the optical constants have been extended to the visual and near-infrared. We highlight that the individual values of k and the absorption cross section depend strongly on the content in transition metals like iron. Based on our measurements, we infer that k values below 10^(-5) are very rare in natural minerals including stardust grains, if they occur at all. Data for k and the absorption cross section are important for various physical properties of stardust grains such as temperature and radiation pressure. With increasing absorption cross section due to impurities, the equilibrium temperature of small grains in circumstellar shells increases as well. We discuss why and to what extent this is the case

Adduct of the blistering warfare agent sesquimustard with human serum albumin and its mass spectrometric identification for biomedical verification of exposure

Apart from the well-known sulfur mustard (SM), additional sulfur-containing blistering chemical warfare agents exist. Sesquimustard (Q) is one of them and five times more blistering than SM. It is a common impurity in mustard mixtures and regularly found in old munitions but can also be used in pure form. Compared to the extensive literature on SM, very little experimental data is available on Q and no protein biomarkers of exposure have been reported. We herein report for the first time the adduct of Q with the nucleophilic Cys34 residue of human serum albumin (HSA) formed in vitro and introduce two novel bioanalytical procedures for detection. After proteolysis of this HSA adduct catalyzed either by pronase or by proteinase K, two biomarkers were identified by high-resolution tandem mass spectrometry (MS/HR MS), namely a dipeptide and a tripeptide, both alkylated at their Cys residue, which we refer to as HETETE-CP and HETETE-CPF. HETETE represents the Q-derived thio-alkyl moiety bearing a terminal hydroxyl group: “hydroxyethylthioethylthioethyl.” Targeting both peptide markers from plasma, a micro liquid chromatography–electrospray ionization tandem mass spectrometry method working in the selected reaction monitoring mode (μLC-ESI MS/MS SRM) was developed and validated as well suited for the verification of exposure to Q. Fulfilling the quality criteria defined by the Organisation for the Prohibition of Chemical Weapons, the novel methods enable the detection of exposure to Q alone or in mixtures with SM. We further report on the relative reactivity of Q compared to SM. Based on experiments making use of partially deuterated Q as the alkylating agent, we rule out a major role for six-membered ring sulfonium ions as relevant reactive species in the alkylation of Cys34. Furthermore, the results of molecular dynamics simulations are indicative that the protein environment around Cys34 allows adduct formation with elongated but not bulky molecules such as Q, and identify important hydrogen bonding interactions and hydrophobic contacts. Graphical abstractSanitätsakademie der Bundeswehr (4256)Peer Reviewe

The Coastal Convective Interactions Experiment (CCIE): understanding the role of sea breezes for hailstorm hotspots in Eastern Australia

The coastal convective interactions experiment (CCIE's) integration of climatological analysis with an intensive field campaign has provided an opportunity for revealing some of the complexities surrounding thunderstorm hotpots in complex physical settings like SEQ. For the CCIE climatological analysis, a continuous 18-yr (July 1997 to June 2015) volumetric reflectivity radar dataset was sourced from the 1.9° S-band weather radar located at Marburg, 50 km west of Brisbane. A cell-based analysis of this archive was performed using a MATLAB implementation of the identification, tracking, and selected analysis algorithms from the Weather Decision Support System-Integrated Information (WDSS-II). The 10-min interval of the Marburg radar volumes creates significant spatial discontinuities between MESH grids from an individual thunderstorm. A preliminary analysis indicates a strong relationship between the presence of the sea breeze and the Boonah hailstorm hotspot, but further analysis is needed to isolate the additional influence of synoptic and topographic drivers. Furthermore, fine-scale field observations of the sea-breeze?thunderstorm interaction events have begun to shed some light on the meteorology of thunderstorm hotspots in South East Queensland (SEQ)

High-resolution NMR structure of an RNA model system: the 14-mer cUUCGg tetraloop hairpin RNA

We present a high-resolution nuclear magnetic resonance (NMR) solution structure of a 14-mer RNA hairpin capped by cUUCGg tetraloop. This short and very stable RNA presents an important model system for the study of RNA structure and dynamics using NMR spectroscopy, molecular dynamics (MD) simulations and RNA force-field development. The extraordinary high precision of the structure (root mean square deviation of 0.3 Å) could be achieved by measuring and incorporating all currently accessible NMR parameters, including distances derived from nuclear Overhauser effect (NOE) intensities, torsion-angle dependent homonuclear and heteronuclear scalar coupling constants, projection-angle-dependent cross-correlated relaxation rates and residual dipolar couplings. The structure calculations were performed with the program CNS using the ARIA setup and protocols. The structure quality was further improved by a final refinement in explicit water using OPLS force field parameters for non-bonded interactions and charges. In addition, the 2′-hydroxyl groups have been assigned and their conformation has been analyzed based on NOE contacts. The structure currently defines a benchmark for the precision and accuracy amenable to RNA structure determination by NMR spectroscopy. Here, we discuss the impact of various NMR restraints on structure quality and discuss in detail the dynamics of this system as previously determined