X-ray structures of the leucine-binding protein illustrate conformational changes and the basis of ligand specificity

The periplasmic leucine-binding protein is the primary receptor for the leucine transport system in Escherichia coli. We report here the structure of an open ligand-free form solved by molecular replacement and refined at 1.5-A resolution. In addition, two closed ligand-bound structures of the same protein are presented, a phenylalanine-bound form at 1.8 A and a leucine-bound structure at a nominal resolution of 2.4 A. These structures show the basis of this protein's ligand specificity, as well as illustrating the conformational changes that are associated with ligand binding. Comparison with earlier structures provides further information about solution conformations, as well as the different specificity of the closely related leucine/isoleucine/valine-binding protein.     

From gene expression analysis to tissue microarrays: a rational approach to identify therapeutic and diagnostic targets in lymphoid malignancies

Mantle cell lymphoma (MCL) is an aggressive lymphoid malignancy for which better treatment strategies are needed. To identify potential diagnostic and therapeutic targets, a signature consisting of MCL-associated genes was selected based on a comprehensive gene expression analysis of malignant and normal B cells. The corresponding protein epitope signature tags were identified and used to raise monospecific, polyclonal antibodies, which were subsequently analyzed on paraffin-embedded sections of malignant and normal tissue. In this study, we demonstrate that the initial selection strategy of MCL-associated genes successfully allows identification of protein antigens either uniquely expressed or overexpressed in MCL compared with normal lymphoid tissues. We propose that genome-based, affinity proteomics, using protein epitope signature tag-induced antibodies, is an efficient way to rapidly identify a number of disease-associated protein candidates of both previously known and unknown identities.     

Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase

Nitric -oxide reductase (NOR) from Paracoccus denitrificans catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N₂O) (2NO + 2H⁺ + 2e⁻ →N₂O + H₂O) by a poorly understood mechanism. NOR contains two low spin hemes c and b, one high spin heme b₃, and a non-heme iron Fe_B. Here, we have studied the reaction between fully reduced NOR and NO using the “flow-flash” technique. Fully (four-electron) reduced NOR is capable of two turnovers with NO. Initial binding of NO to reduced heme b₃ occurs with a time constant of ∼1 μs at 1.5 mm NO, in agreement with earlier studies. This reaction is [NO]-dependent, ruling out an obligatory binding of NO to Fe_B before ligation to heme b₃. Oxidation of hemes b and c occurs in a biphasic reaction with rate constants of 50 s⁻¹ and 3 s⁻¹ at 1.5 mm NO and pH 7.5. Interestingly, this oxidation is accelerated as [NO] is lowered; the rate constants are 120 s⁻¹ and 12 s⁻¹ at 75 μm NO. Protons are taken up from solution concomitantly with oxidation of the low spin hemes, leading to an acceleration at low pH. This effect is, however, counteracted by a larger degree of substrate inhibition at low pH. Our data thus show that substrate inhibition in NOR, previously observed during multiple turnovers, already occurs during a single oxidative cycle. Thus, NO must bind to its inhibitory site before electrons redistribute to the active site. The further implications of our data for the mechanism of NO reduction by NOR are discussed.     

Effects from shear stress on morphology and growth of early stages of Norway spruce somatic embryos

The shear stress effect on directional expansion of pro embryogenic masses (PEMs) and suspensor cell development of somatic embryos of Norway spruce (Picea abies) at the proliferation stage was studied by a direct and quantitative image analysis system. The experimental system allowed for detailed observations of the effect of hydrodynamic shear stress in rotating and deforming liquid cultures of proliferating Norway spruce somatic embryos. Briefly, somatic embryos at an early development stage comprised only of clusters of meristematic cells without suspensor cells were fixed on an alginate film. The alginate film was affixed on the bottom of a flow cell and the somatic embryos were subjected to laminar flow through the chamber of the flow cell. Magnified images of the cell clusters were collected every 24 h. The image data was processed based on a normalized cross‐correlation method, capable of measuring morphological and size features of individual cell clusters in both temporal and spatial domains. No suspensor cells developed in the cell clusters under shear stress of 140 s^?1 for the duration of the experiments. Cell clusters in the control cultured in stationary liquid conditions developed suspensor cells after 5–9 days in culture. Furthermore, the radial growth of meristematic cell clusters was inhibited by shear rates of 86 and 140 s^?1, corresponding to shear stress of 0.086 and 0.14 N/m², compared to growth under stationary conditions. The shear rate showed a significant negative correlation to growth rate. Control group showed no preference for direction during growth under static conditions. Biotechnol. Bioeng. 2010; 105: 588–599. © 2009 Wiley Periodicals, Inc.     

Transmembrane topology of FRO2, a ferric chelate reductase from Arabidopsis thaliana

Iron uptake in Arabidopsis thaliana is mediated by ferric chelate reductase FRO2, a transmembrane protein belonging to the flavocytochrome b family. There is no high resolution structural information available for any member of this family. We have determined the transmembrane topology of FRO2 experimentally using the alkaline phosphatase fusion method. The resulting topology is different from that obtained by theoretical predictions and contains 8 transmembrane helices, 4 of which build up the highly conserved core of the protein. This core is present in the entire flavocytochrome b family. The large water soluble domain of FRO2, which contains NADPH, FAD and oxidoreductase sequence motifs, was located on the inside of the membrane.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at This work was supported by grants from the Swedish Research Council (VR) to S. Al-Karadaghi and Hans Hebert.     

Metabolic profiles and genetic diversity of denitrifying communities in activated sludge after addition of methanol or ethanol

External carbon sources can enhance denitrification rates and thus improve nitrogen removal in wastewater treatment plants. The effects of adding methanol and ethanol on the genetic and metabolic diversity of denitrifying communities in activated sludge were compared using a pilot-scale plant with two parallel lines. A full-scale plant receiving the same municipal wastewater, but without external carbon source addition, was the reference. Metabolic profiles obtained from potential denitrification rates with 10 electron donors showed that the denitrifying communities altered their preferences for certain compounds after supplementation with methanol or ethanol and that methanol had the greater impact. Clone libraries of nirK and nirS genes, encoding the two different nitrite reductases in denitrifiers, revealed that methanol also increased the diversity of denitrifiers of the nirS type, which indicates that denitrifiers favored by methanol were on the rise in the community. This suggests that there might be a niche differentiation between nirS and nirK genotypes during activated sludge processes. The composition of nirS genotypes also varied greatly among all samples, whereas the nirK communities were more stable. The latter was confirmed by denaturing gradient gel electrophoresis of nirK communities on all sampling occasions. Our results support earlier hypotheses that the compositions of denitrifier communities change during predenitrification processes when external carbon sources are added, although no severe effect could be observed from an operational point of view.     

Thermofluor-based high-throughput stability optimization of proteins for structural studies

Production of proteins well suited for structural studies is inherently difficult and time-consuming. Protein sample homogeneity, stability, and solubility are strongly correlated with the proteins' probability of yielding crystals, and optimization of these properties will improve success rates of crystallization. In the current study, we applied the thermofluor method as a high-throughput approach for identifying optimal protein formulation for crystallization. The method also allowed optimal stabilizing buffer compositions to be rapidly identified for each protein. Furthermore, the method allowed the identification of potential ligands, physiological or non-physiological, that can be used in subsequent crystallization trials. For this study, the thermally induced melting points were determined in different buffers as well as with additives for a total of 25 Escherichia coli proteins. Crystallization trials were set up together with stabilizing and destabilizing additives identified using thermofluor screening. A twofold increase in the number of crystallization leads was observed when the proteins were cocrystallized with stabilizing additives as compared with experiments without these additives. This suggests that thermofluor constitutes an efficient generic high-throughput method for identification of protein properties predictive of crystallizability.     

Improved variance estimation of classification performance via reduction of bias caused by small sample size

Background  

Supervised learning for classification of cancer employs a set of design examples to learn how to discriminate between tumors. In practice it is crucial to confirm that the classifier is robust with good generalization performance to new examples, or at least that it performs better than random guessing. A suggested alternative is to obtain a confidence interval of the error rate using repeated design and test sets selected from available examples. However, it is known that even in the ideal situation of repeated designs and tests with completely novel samples in each cycle, a small test set size leads to a large bias in the estimate of the true variance between design sets. Therefore different methods for small sample performance estimation such as a recently proposed procedure called Repeated Random Sampling (RSS) is also expected to result in heavily biased estimates, which in turn translates into biased confidence intervals. Here we explore such biases and develop a refined algorithm called Repeated Independent Design and Test (RIDT).     

Influence of chronic and acute spinal cord injury on skeletal muscle Na+-K+-ATPase and phospholemman expression in humans

Na(+)-K(+)-ATPase is an integral membrane protein crucial for the maintenance of ion homeostasis and skeletal muscle contractibility. Skeletal muscle Na(+)-K(+)-ATPase content displays remarkable plasticity in response to long-term increase in physiological demand, such as exercise training. However, the adaptations in Na(+)-K(+)-ATPase function in response to a suddenly decreased and/or habitually low level of physical activity, especially after a spinal cord injury (SCI), are incompletely known. We tested the hypothesis that skeletal muscle content of Na(+)-K(+)-ATPase and the associated regulatory proteins from the FXYD family is altered in SCI patients in a manner dependent on the severity of the spinal cord lesion and postinjury level of physical activity. Three different groups were studied: 1) six subjects with chronic complete cervical SCI, 2) seven subjects with acute, complete cervical SCI, and 3) six subjects with acute, incomplete cervical SCI. The individuals in groups 2 and 3 were studied at months 1, 3, and 12 postinjury, whereas individuals with chronic SCI were compared with an able-bodied control group. Chronic complete SCI was associated with a marked decrease in [(3)H]ouabain binding site concentration in skeletal muscle as well as reduced protein content of the α(1)-, α(2)-, and β(1)-subunit of the Na(+)-K(+)-ATPase. In line with this finding, expression of the Na(+)-K(+)-ATPase α(1)- and α(2)-subunits progressively decreased during the first year after complete but not after incomplete SCI. The expression of the regulatory protein phospholemman (PLM or FXYD1) was attenuated after complete, but not incomplete, cervical SCI. In contrast, FXYD5 was substantially upregulated in patients with complete SCI. In conclusion, the severity of the spinal cord lesion and the level of postinjury physical activity in patients with SCI are important factors controlling the expression of Na(+)-K(+)-ATPase and its regulatory proteins PLM and FXYD5.