Structural and functional roles of a conserved proline residue in the alpha2 helix of Escherichia coli thioredoxin

Proline 40 in Escherichia coli thioredoxin is located close to the redox active site (Cys32-Cys35) within the alpha2 helix. The conservation of this residue among most of the thioredoxins suggests that it could play an important role in the structure and/or function of this protein. We have substituted Pro40 for Ala by using site- directed mutagenesis and expressed the mutant P40A in E.coli. The effects of the mutation on the biophysical and biological properties of thioredoxin have been analyzed and compared with molecular dynamics simulations. Modeling predicted that the replacement of Pro40 by Ala induced a displacement of the active site which exposes Trp31 to the solvent and opens a cleft located between helices alpha2 and alpha3. The solvation free energy (SFE) calculation also indicated that P40A became more hydrophobic as W31 became more accessible. These predictions were totally in agreement with the experimental results. The mutant P40A exhibited chromatographic behavior and fluorescence properties very different from those of the wild-type (WT) protein, in relationship with the displacement of W31. The determination of the free energy of unfolding of P40A showed that the mutant was globally destabilized by 2.9 kcal/mol. However, the effect of the mutation on the transition curve was highly unusual as the midpoint of the unfolding transition increased, indicating that some local structures were actually stabilized by the mutation. Despite these structural modifications, neither the ability of the protein to reduce a chloroplastic enzyme nor its reactivity with the bacterial reductase decreased. The only functional difference was the higher stability of P40A in light activation of NADP-malate dehydrogenase under air, which suggests that the mutant was less rapidly re-oxidized than WT. Therefore, it can be concluded that Pro40 is not essential for maintaining the redox function of thioredoxin but rather is required for the stability of the protein.      

Neural nets – types,configurations and pitfalls

Fuzzy logic, neural nets and genetic algorithms form the core of soft computing methods. They are useful when there is no possibility to compute an exact mathematical model (hard computing). Neural nets have the ability to learn by example. This advantage is exploited by a lot of applications and many software packages make it quite easy to use neural nets. A stage is reached, where some critical remarks should be made in order to avoid disappointments. Some frequently used net types (backpropagation, LVQ, SOM) are discussed together with configuration and training problems. Important topics are the avoidance of under- and overfit and the remark that neural nets produce correct outputs only if the inputs lie in the part of the feature space, the net was trained for. Therefore, a detailed analysis of the training data set should be made. In the context of safety relevant applications the missing interpretability of neural net outputs is often criticized. Fuzzy-neuro-systems try to improve this situation.     

Smooth shapes with spherical topology: Beyond traditional modeling,efficient deformation,and interaction

Computational Visual Media - Existing shape models with spherical topology are typically designed either in the discrete domain using interpolating polygon meshes or in the continuous domain using...     

New light stabilizer for coextruded polycarbonate sheet

Weatherable polycarbonate sheet can be produced by coextruding a cap layer containing a UV absorber over a minimally stabilized bulk layer. A new UV absorber based on hydroxyphenyltriazine chemistry has been developed that is especially suited to this application. The product provides superior long term weatherability because of its strong UV absorbance and its excellent photostability. It is also relatively nonvolatile and has only a minimal effect on melt viscosity.     

Germany’s journey toward 14 Tesla human magnetic resonance

Multiple sites within Germany operate human MRI systems with magnetic fields either at 7 Tesla or 9.4 Tesla. In 2013, these sites formed a network to facilitate and harmonize the research being conducted at the different sites and make this technology available to a larger community of researchers and clinicians not only within Germany, but also worldwide. The German Ultrahigh Field Imaging (GUFI) network has defined a strategic goal to establish a 14 Tesla whole-body human MRI system as a national research resource in Germany as the next progression in magnetic field strength. This paper summarizes the history of this initiative, the current status, the motivation for pursuing MR imaging and spectroscopy at such a high magnetic field strength, and the technical and funding challenges involved. It focuses on the scientific and science policy process from the perspective in Germany, and is not intended to be a comprehensive systematic review of the benefits and technical challenges of higher field strengths.

     

Introduction to a new class of high performance light stabilizers and the influence of light stabilizers structure on the polymer's life time

The mode of action of two classes of UV absorbers (2-(hydroxyphenyl)benzotriazoles and 2-(2′-hydroxyphenyl)-1,3,5-triazines) is discussed. Theoretical explanations for the superior light stability of the latter are given and confirmed by measured half life times in polycarbonate and solution. A mathematical model for the quantitative prediction of light protection factors is proposed.     

Valuing flexibilities in the design of urban water management systems

Climate change and rapid urbanization requires decision-makers to develop a long-term forward assessment on sustainable urban water management projects. This is further complicated by the difficulties of assessing sustainable designs and various design scenarios from an economic standpoint. A conventional valuation approach for urban water management projects, like Discounted Cash Flow (DCF) analysis, fails to incorporate uncertainties, such as amount of rainfall, unit cost of water, and other uncertainties associated with future changes in technological domains. Such approach also fails to include the value of flexibility, which enables managers to adapt and reconfigure systems over time as uncertainty unfolds.     

The N-terminal half of initiation factor IF3 is folded as a stable independent domain

Initiation factor IF3 plays an essential role in the initiation of protein translation by binding to the 30S ribosomal subunit and selecting a proper tRNA(fMet)/initiation codon complex. The domain structure of IF3 from Escherichia coli has been investigated by limited proteolysis followed by mass spectrometry and protein sequencing of the resulting peptides. This analysis revealed a highly segmented structure with two independent domains connected by a charged linker peptide, highly susceptible to proteolytic cleavage. The N-terminal domain is very stable and comparison of its 2-D NMR spectrum with that of intact IF3 revealed that it retains its three-dimensional fold.     

Structural and functional roles of a conserved proline residue in the alpha2 helix of Escherichia coli thioredoxin

Proline 40 in Escherichia coli thioredoxin is located close to the redox active site (Cys32-Cys35) within the alpha2 helix. The conservation of this residue among most of the thioredoxins suggests that it could play an important role in the structure and/or function of this protein. We have substituted Pro40 for Ala by using site-directed mutagenesis and expressed the mutant P40A in E.coli. The effects of the mutation on the biophysical and biological properties of thioredoxin have been analyzed and compared with molecular dynamics simulations. Modeling predicted that the replacement of Pro40 by Ala induced a displacement of the active site which exposes Trp31 to the solvent and opens a cleft located between helices alpha2 and alpha3. The solvation free energy (SFE) calculation also indicated that P40A became more hydrophobic as W31 became more accessible. These predictions were totally in agreement with the experimental results. The mutant P40A exhibited chromatographic behavior and fluorescence properties very different from those of the wild-type (WT) protein, in relationship with the displacement of W31. The determination of the free energy of unfolding of P40A showed that the mutant was globally destabilized by 2.9 kcal/mol. However, the effect of the mutation on the transition curve was highly unusual as the midpoint of the unfolding transition increased, indicating that some local structures were actually stabilized by the mutation. Despite these structural modifications, neither the ability of the protein to reduce a chloroplastic enzyme nor its reactivity with the bacterial reductase decreased. The only functional difference was the higher stability of P40A in light activation of NADP-malate dehydrogenase under air, which suggests that the mutant was less rapidly re-oxidized than WT. Therefore, it can be concluded that Pro40 is not essential for maintaining the redox function of thioredoxin but rather is required for the stability of the protein.     

Identification of surface-associated proteins in the probiotic bacterium Lactobacillus rhamnosus GG

Six methods for extraction of surface-associated proteins were applied to Lactobacillus rhamnosus GG (LGG) cells harvested at early stationary phase. The six separation profiles of proteins analysed by gel electrophoresis were similar but markedly different from total protein extracts, suggesting that those profiles contained surface-associated proteins and were not contaminated by cytoplasmic proteins. Among the methods tested, treatment with 5 m LiCl appeared to be the most suitable for surface-associated protein extraction in this probiotic strain, since it allowed a maximum recovery of proteins. Among the proteins identified, ribosomal proteins, cytoplasmic proteins already described on the bacterial surface, and several proteins predicted to be surface-associated were identified by tandem mass spectrometry. The pH of the buffer affected the efficiency of protein extraction, with less surface proteins being extracted from LGG cells at acidic pH. Some of the surface proteins identified in this work may help explain some of the probiotic traits of LGG, notably these concerning the interaction of LGG strain with the human host.