Combined Mutagenesis and Kinetics Characterization of the Bilin‐Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803

The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red‐absorbing parental and a green‐absorbing photoproduct state (λ_max=649 and 536 nm, respectively). Conversions in both directions were followed by time‐resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 μs, 390 μs, and 1.5 ms) for the red‐to‐green conversion, and 1.2 μs, 340 μs, and 1 ms for the green‐to‐red conversion. In addition to the wild‐type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site‐directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp. PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red‐absorbing, WT‐like protein. Irradiation, however, not only converted it into the green‐absorbing form, but also produced a 660 nm, further‐red‐shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.     

Pneumocandin Biosynthesis: Involvement of a trans‐Selective Proline Hydroxylase

Echinocandins are cyclic nonribosomal hexapeptides based mostly on nonproteinogenic amino acids and displaying strong antifungal activity. Despite previous studies on their biosynthesis by fungi, the origin of three amino acids, trans‐4‐ and trans‐3‐hydroxyproline, as well as trans‐3‐hydroxy‐4‐methylproline, is still unknown. Here we describe the identification, overexpression, and characterization of GloF, the first eukaryotic α‐ketoglutarate/Fe^II‐dependent proline hydroxylase from the pneumocandin biosynthesis cluster of the fungus Glarea lozoyensis ATCC 74030. In in vitro transformations with L ‐proline, GloF generates trans‐4‐ and trans‐3‐hydroxyproline simultaneously in a ratio of 8:1; the latter reaction was previously unknown for proline hydroxylase catalysis. trans‐4‐Methyl‐L ‐proline is converted into the corresponding trans‐3‐hydroxyproline. All three hydroxyprolines required for the biosynthesis of the echinocandins pneumocandins A₀ and B₀ in G. lozoyensis are thus provided by GloF. Sequence analyses revealed that GloF is not related to bacterial proline hydroxylases, and none of the putative proteins with high sequence similarity in the databases has been characterized so far.     

High‐Affinity Carbohydrate Binding by Trimeric Benzoboroxoles Measured on Carbohydrate Arrays

Carbohydrates are involved in a wide range of biological processes of pharmaceutical relevance. The selective recognition of carbohydrates is therefore of great interest in biology and medicine. In this study we present the synthesis of fluorescent multimeric benzoboroxoles and the analysis of multivalent binding processes to immobilized carbohydrate arrays by fluorescence spectroscopy. We observed high binding affinities of trimeric benzoboroxoles by determination of K_Dsurf values for their interaction with α‐Gal on glass chips. The observed K_Dsurf values were in the mid‐nM range (49 and 104 nM ) and are comparable to the K_Dsurf values for binding of natural lectins, such as that of ConA to immobilized α‐Man (79 nM ). The array technology was found to be an excellent tool for studying the binding processes of multivalent lectin mimetics with respect to profiling and quantitation.     

Magic Angle Spinning NMR of Macromolecular and Supramolecular Systems

Sideband patterns in single quantum and double quantum magic angle spinning NMR spectra are reviewed. Their use in elucidating non-covalent interactions, such as hydrogen bonds or pi-pi stacking, is demonstrated by studies of supramolecular systems of current interest. Moreover, the effects of local and collective motions are illustrated by examples from supramolecular and macromolecular science.     

Investigation on the effectiveness of the repair method 8.3 “Corrosion protection by increasing the electrical resistivity” in chloride-containing concrete Part 2: Chloride redistribution in concrete after application of a system sealing surface protective coating

This DFG-funded research project aimed to gain a better understanding of the mechanisms of the W-Cl repair principle within the framework of fundamental investigations, to contribute to the creation of the necessary basis for a broader application of the repair principle in practice. The focus was on the development of a model to describe the chloride redistribution after the application of a system sealing surface protective coating. On the basis of Fick's second law of diffusion, a mathematical model with a self-contained analytical solution was developed, with the help of which the chloride redistribution after application of a system sealing surface protective coating can be calculated under the idealized assumption of complete water saturation of the concrete. Furthermore, the influence of the dehydration of the concrete, expected as a result of the application of the repair principle W-Cl, on the chloride redistribution was investigated. On the basis of laboratory tests and numerical simulations, material-specific reduction functions were developed to quantify the relationship between the chloride diffusion coefficient and the ambient humidity.     

ZF公司面向中国的高性能变速器

文章介绍了ZF公司为轮式装载机特别研发的WG系列高性能变速器,其产品系列可覆盖9-30吨级的车辆.ZF高性能的动力换档变速器具有简单和坚固的零部件设计、采用精确调整轴承、整体式壳体结构和优越的电控系统等优点.     

大型X射线光学仪器:单层镜和多层镜的制造与表征

150~500 mm长度的各种X射线光学元件可用于光束导引,光束调整,以及单色化。本文介绍了两种不同的大型X射线反射镜。第一种为单层反射镜,这种反射镜以2°掠入射角在软X射线区(50~200 eV)起全反射镜作用,可用于自由电子激光器,如德国汉堡的FLASH。第二种是多层镜,由于它的布喇格反射特性,适于作为反射镜以0.4~1°的入射角用于硬X射线区(20~50 keV),如层析光束线的同步辐射存储环中。两种反射镜都用最新物理汽相淀积法制备,并用磁控溅射来实现X射线光学应用所需要的优良光学品质。这一淀积工艺使不同批次的镀膜稳定性良好,有利于实际反射镜在优质衬底上的最后淀积。单层镜和多层镜在它们的相关能量范围内都有很高的反射率,表面粗糙度也很低,且在整个光学波长区这些特性表现均匀。文中所叙相关研究都是借助X射线反射计量(XRR)法,透射电子显微镜(TEM)、光学轮廓仪(OP),以及原子力显微镜(AFM)完成的。     

Nondestructive and noncontact method for determining the spring constant of rectangular cantilevers

We present here an experimental setup and suggest an extension to the long existing added-mass method for the calibration of the spring constant of atomic force microscope cantilevers. Instead of measuring the resonance frequency shift that results from attaching particles of known masses to the end of cantilevers, we load them with water microdrops generated by a commercial inkjet dispenser. Such a device is capable of generating drops, and thus masses, of extremely reproducible size. This makes it an ideal tool for calibration tasks. Moreover, the major advantage of water microdrops is that they allow for a nearly contactless calibration: no mechanical micromanipulation of particles on cantilevers is required, neither for their deposition nor for removal. After some seconds the water drop is completely evaporated, and no residues are left on the cantilever surface or tip. We present two variants: we vary the size of the drops and deposit them at the free end of the cantilever, or we keep the size of the drops constant and vary their position along the cantilever. For the second variant, we implemented also numerical simulations. Spring constants measured by this method are comparable to results obtained by the thermal noise method, as we demonstrate for six different cantilevers.     

An apparatus for in situ spectroscopy of radiation damage of polymers by bombardment with high-energy heavy ions

A new target station providing Fourier transform infrared (FT-IR) spectroscopy and residual gas analysis (RGA) for in situ observation of ion-induced changes in polymers has been installed at the GSI Helmholtz Centre for Heavy Ion Research. The installations as well as first in situ measurements at room temperature are presented here. A foil of polyimide Kapton HN(?) was irradiated with 1.1 GeV Au ions. During irradiation several in situ FT-IR spectra were recorded. Simultaneously outgassing degradation products were detected with the RGA. In the IR spectra nearly all bands decrease due to the degradation of the molecular structure. In the region from 3000 to 2700 cm(-1) vibration bands of saturated hydrocarbons not reported in literature so far became visible. The outgassing experiments show a mixture of C(2)H(4), CO, and N(2) as the main outgassing components of polyimide. The ability to combine both analytical methods and the opportunity to measure a whole fluence series within a single experiment show the efficiency of the new setup.