Neue Medien in der Bildung – technische oder kulturelle Herausforderung? (Zwischen-)Bericht aus der Projektpraxis ePUSH

Die Strategien zur Förderung von E-Learning an Universitäten haben sich seit der Entwicklung des World Wide Web in den 90er Jahren stetig verändert. Betrachtet man die Entwicklung der Förderlinien, so lässt sich ein allmählicher Wandel der Zielsetzungen erkennen. Dieser Wandel der Zielsetzungen deutet darauf hin, dass mit der Entwicklung des Internet als neuem Verbreitungsmedium kulturelle Entwicklungen einhergehen, die sich im Kern auf die Bedeutung und die Organisation von Universitäten auswirken. Dieser Prozess wird am Beispiel des Hochschulentwicklungsprojekts ePUSH der Fakultät für Erziehungswissenschaft, Psychologie und Bewegungswissenschaft an der Universität Hamburg verdeutlicht. (DIPF/ Orig.

Probing the urea dependence of residual structure in denatured human α-lactalbumin

Backbone 15N relaxation parameters and 15N–1HN residual dipolar couplings (RDCs) have been measured for a variant of human α-lactalbumin (α-LA) in 4, 6, 8 and 10 M urea. In the α-LA variant, the eight cysteine residues in the protein have been replaced by alanines (all-Ala α-LA). This protein is a partially folded molten globule at pH 2 and has been shown previously to unfold in a stepwise non-cooperative manner on the addition of urea. 15N R2 values in some regions of all-Ala α-LA show significant exchange broadening which is reduced as the urea concentration is increased. Experimental RDC data are compared with RDCs predicted from a statistical coil model and with bulkiness, average area buried upon folding and hydrophobicity profiles in order to identify regions of non-random structure. Residues in the regions corresponding to the B, D and C-terminal 310 helices in native α-LA show R2 values and RDC data consistent with some non-random structural propensities even at high urea concentrations. Indeed, for residues 101–106 the residual structure persists in 10 M urea and the RDC data suggest that this might include the formation of a turn-like structure. The data presented here allow a detailed characterization of the non-cooperative unfolding of all-Ala α-LA at higher concentrations of denaturant and complement previous studies which focused on structural features of the molten globule which is populated at lower concentrations of denaturant

A refined solution structure of hen lysozyme determined using residual dipolar coupling data

A high resolution NMR structure of hen lysozyme has been determined using 209 residual 1H–15N dipolar coupling restraints from measurements made in two different dilute liquid crystalline phases (bicelles) in conjunction with a data set of 1632 NOE distance restraints, 110 torsion angle restraints, and 60 hydrogen bond restraints. The ensemble of 50 low-energy calculated structures has an average backbone RMSD of 0.50±0.13Å to the mean structure and of 1.49±0.10Å to the crystal structure of hen lysozyme. To assess the importance of the dipolar coupling data in the structure determination, the final structures are compared with an ensemble calculated using an identical protocol but excluding the dipolar coupling restraints. The comparison shows that structures calculated with the dipolar coupling data are more similar to the crystal structure than those calculated without, and have better stereochemical quality. The structures also show improved quality factors when compared with additional dipolar coupling data that were not included in the structure calculations, with orientation-dependent 15N chemical shift changes measured in the bicelle solutions, and with T1/T2 values obtained from 15N relaxation measurements. Analysis of the ensemble of NMR structures and comparisons with crystal structures, 15N relaxation data, and molecular dynamics simulations of hen lysozyme provides a detailed description of the solution structure of this protein and insights into its dynamical behavior

Modular, triple-resonance, transmission line DNP MAS probe for 500 MHz/330 GHz

© 2019 Elsevier Inc. We describe the design and construction of a modular, triple-resonance, fully balanced, DNP-MAS probe based on transmission line technology and its integration into a 500 MHz/330 GHz DNP-NMR spectrometer. A novel quantitative probe design and characterization strategy is developed and employed to achieve optimal sensitivity, RF homogeneity and excellent isolation between channels. The resulting three channel HCN probe has a modular design with each individual, swappable module being equipped with connectorized, transmission line ports. This strategy permits attachment of a mating connector that facilitates accurate impedance measurements at these ports and allows characterization and adjustment (e.g. for balancing or tuning/matching) of each component individually. The RF performance of the probe is excellent; for example, the 13C channel attains a Rabi frequency of 280 kHz for a 3.2 mm rotor. In addition, a frequency tunable 330 GHz gyrotron operating at the second harmonic of the electron cyclotron frequency was developed for DNP applications. Careful alignment of the corrugated waveguide led to minimal loss of the microwave power, and an enhancement factor ε = 180 was achieved for U-13C urea in the glassy matrix at 80 K. We demonstrated the operation of the system with acquisition of multidimensional spectra of cross-linked lysozyme crystals which are insoluble in glycerol-water mixtures used for DNP and samples of RNA

NMR Structural Profiling of Transcriptional Intermediates Reveals Riboswitch Regulation by Metastable RNA Conformations

Gene repression induced by the formation of transcriptional terminators represents a prime example for the coupling of RNA synthesis, folding, and regulation. In this context, mapping the changes in available conformational space of transcription intermediates during RNA synthesis is important to understand riboswitch function. A majority of riboswitches, an important class of small metabolite-sensing regulatory RNAs, act as transcriptional regulators, but the dependence of ligand binding and the subsequent allosteric conformational switch on mRNA transcript length has not yet been investigated. We show a strict fine-tuning of binding and sequence-dependent alterations of conformational space by structural analysis of all relevant transcription intermediates at single-nucleotide resolution for the I-A type 2′dG-sensing riboswitch from <i>Mesoplasma florum</i> by NMR spectroscopy. Our results provide a general framework to dissect the coupling of synthesis and folding essential for riboswitch function, revealing the importance of metastable states for RNA-based gene regulation

Noncovalent Spin Labeling of Riboswitch RNAs To Obtain Long-Range Structural NMR Restraints

Paramagnetic relaxation enhancement (PRE) NMR is a powerful method to study structure, dynamics and function of proteins. Up to now, the application of PRE NMR on RNAs is a significant challenge due to the limited size of chemically synthesized RNA. Here, we present a noncovalent spin labeling strategy to spin label RNAs in high yields required for NMR studies. The approach requires the presence of a helix segment composed of about 10 nucleotides (nt) but is not restricted by the size of the RNA. We show successful application of this strategy on the 2′dG sensing aptamer domain of <i>Mesoplasma florum</i> (78 nt). The aptamer domain was prepared in two fragments. A larger fragment was obtained by biochemical means, while a short spin labeled fragment was prepared by chemical solid-phase synthesis. The two fragments were annealed noncovalently by hybridization. We performed NMR, cw-EPR experiments and gel shift assays to investigate the stability of the two-fragment complex. NMR analysis in ¹⁵N-TROSY and ¹H,¹H-NOESY spectra of both unmodified and spin labeled aptamer domain show that the fragmented system forms a stable hybridization product, is in structural agreement with the full length aptamer domain and maintains its function. Together with structure modeling, experimentally determined ¹H-Γ₂ rates are in agreement with reported crystal structure data and show that distance restraints up to 25 Å can be obtained from NMR PRE data of RNA