Exploring the open pore of the potassium channel from Streptomyces lividans

The tetrameric potassium channel from Streptomyces lividans (KcsA) embedded in planar bilayers exhibits the following electrophysiological characteristics: (i) K+ ions can cross the pore in a highly hydrated state (nH2O > or = 6), (ii) the selectivity for K+ exceeds that for Na+ ions by 11 times, and both Ca2+ and Mg2+ are permeant, (iii) the internal side is blocked by Ba2+ ions in a voltage-dependent manner, (iv) intrinsic rectification is due to gating, depending on the direction of the electric field, (v) the internal side is pH-sensitive, and (vi) the open pore has a diameter of approximately 5.8 A. In conclusion, our results show that ion conduction and selectivity of KcsA cannot easily be reconciled with the properties deduced from the rigid crystal structure [Doyle et al., Science 280 (1998) 69-77], which must be concluded to have the pore trapped in its closed state.     

Chromosomal promoter replacement in Saccharomyces cerevisiae: Construction of conditional lethal strains for the cloning of glycosyltransferases from various organisms

Heterologous complementation in yeast has been a successful tool for cloning and characterisation of genes from various organisms. Therefore we constructed conditionally lethal Saccharomyces cerevisiae strains by replacing the endogenous promoter from the genes of interest (glycosyltransferases) by the stringently regulated GAL1-promoter, by a technique called chromosomal promoter replacement. Such yeast strains were constructed for the genes Alg 1, Alg7, Sec59, Wbp1 involved in N-Glycosylation, the genes Gpi2, Gpi3/Spt14, Gaal, Pis1, involved in GPI-anchor biosynthesis and Dpm involved in both pathways. All strains show the expected conditionally lethal phenotype on glucose-containing medium when expression of the respective gene is turned off.     

d-matrix – database exploration,visualization and analysis

Background  

Motivated by a biomedical database set up by our group, we aimed to develop a generic database front-end with embedded knowledge discovery and analysis features. A major focus was the human-oriented representation of the data and the enabling of a closed circle of data query, exploration, visualization and analysis.     

Compromised influenza virus-specific CD8(+)-T-cell memory in CD4(+)-T-cell-deficient mice

The primary influenza A virus-specific CD8(+)-T-cell responses measured by tetramer staining of spleen, lymph node, and bronchoalveolar lavage (BAL) lymphocyte populations were similar in magnitude for conventional I-A(b+/+) and CD4(+)-T-cell-deficient I-A(b-/-) mice. Comparable levels of virus-specific cytotoxic-T-lymphocyte activity were detected in the inflammatory exudate recovered by BAL following challenge. However, both the size of the memory T-cell pool and the magnitude of the recall response in the lymphoid tissues (but not the BAL specimens) were significantly diminished in mice lacking the CD4(+) subset. Also, the rate of virus elimination from the infected respiratory tract slowed at low virus loads following challenge of na?ve and previously immunized I-A(b-/-) mice. Thus, though the capacity to mediate the CD8(+)-T-cell effector function is broadly preserved in the absence of concurrent CD4(+)-T-cell help, both the maintenance and recall of memory are compromised and the clearance of residual virus is delayed. These findings are consistent with mathematical models that predict virus-host dynamics in this, and other, models of infection.     

The consensus concept for thermostability engineering of proteins: further proof of concept

Previously, we calculated a consensus amino acid sequence from 13 homologous fungal phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15-26 degrees C more thermostable than all parent phytases used in its design [Lehmann et al. (2000)Protein Eng., 13, 49-57]. In the present study, inclusion of six further phytase sequences in the amino acid sequence alignment resulted in the replacement of 38 amino acid residues in either one or both of the new consensus phytases-10 and -11. Since consensus phytase-10, again, was 7.4 degrees C more thermostable than consensus phytase-1, the thermostability effects of most of the 38 amino acid substitutions were tested by site-directed mutagenesis. Both stabilizing and destabilizing mutations were identified, but all affected the stability of the enzyme by <3 degrees C. The combination of all stabilizing amino acid exchanges in a multiple mutant of consensus phytase-1 increased the unfolding temperature from 78.0 to 88.5 degrees C. Likewise, back-mutation of four destabilizing amino acids and introduction of an additional stabilizing amino acid in consensus phytase-10 further increased the unfolding temperature from 85.4 to 90.4 degrees C. The thermostabilization achieved is the result of a combination of slight improvements from multiple amino acid exchanges rather than being the effect of a single or of just a few dominating mutations that have been introduced by chance. The present findings support the general validity of the consensus concept for thermostability engineering of proteins.     

Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework

Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells. These so-called intrabodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or by blocking interactions with other proteins. However, the intracellular expression and activity of many single-chain Fvs are limited by their instability and folding efficiency in the reducing intracellular environment, where the highly conserved intrachain disulfide bonds do not form. In the present work, we used an in vivo selection system to isolate novel antigen-binding intrabodies. We screened two intrabody libraries carrying a randomized third hypervariable loop onto the heavy chain of a stable framework, which had been further optimized by random mutagenesis for better behavior in the selection system, and we biophysically characterized the selected variants to interpret the outcome of the selection. Our results show that single-framework intrabody libraries can be directly screened in vivo to rapidly select antigen-specific intrabodies.     

Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants

Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting cis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter “calcoaceticus” ADP1. We show that they transform, in addition to cis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis,cis-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three-dimensional structures, variants of P. putida muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate conversion. The rate of 2-chloro-cis,cis-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other.     

Quantitative analysis of the local rates of growth of dicot leaves at a high temporal and spatial resolution,using image sequence analysis

A new technique is presented for quantitative mapping of dicot leaf growth at high spatial and temporal resolution, at a speed making online-mapping feasible. Time lapse video sequences of growing leaves are captured by a personal computer (PC) with a frame-grabber board and a standard CCD camera, and evaluated using algorithms that have been recently developed to analyse motion in dynamic image sequences. Growth can be detected at under 1% per hour, with a time resolution of minutes and a spatial resolution of a few millimeters. The new technique has been verified by comparing it with classical approaches to map integrated growth. Diurnal courses of leaf growth of Ricinus communis and tobacco are presented to demonstrate the localised character of growth in leaves. Expansion growth is restricted to the base of the leaf and is restricted to a few hours at the end of the night and the start of the day. The high resolution of the method is illustrated by mapping the responses to step-changes in leaf turgor. A 3 bar turgor jump led to a rapid transient expansion over the entire length of the leaf that was partially reversed when the turgor was relaxed.     

A Single Amino Acid Tunes Ca2+ Inhibition of Brain Liver Intestine Na+ Channel (BLINaC)

Ion channels of the degenerin/epithelial Na⁺ channel gene family are Na⁺ channels that are blocked by the diuretic amiloride and are implicated in several human diseases. The brain liver intestine Na⁺ channel (BLINaC) is an ion channel of the degenerin/epithelial Na⁺ channel gene family with unknown function. In rodents, it is expressed mainly in brain, liver, and intestine, and to a lesser extent in kidney and lung. Expression of rat BLINaC (rBLINaC) in Xenopus oocytes leads to small unselective currents that are only weakly sensitive to amiloride. Here, we show that rBLINaC is inhibited by micromolar concentrations of extracellular Ca²⁺. Removal of Ca²⁺ leads to robust currents and increases Na⁺ selectivity of the ion pore. Strikingly, the species ortholog from mouse (mBLINaC) has an almost 250-fold lower Ca²⁺ affinity than rBLINaC, rendering mBLINaC constitutively active at physiological concentrations of extracellular Ca²⁺. In addition, mBLINaC is more selective for Na⁺ and has a 700-fold higher amiloride affinity than rBLINaC. We show that a single amino acid in the extracellular domain determines these profound species differences. Collectively, our results suggest that rBLINaC is opened by an unknown ligand whereas mBLINaC is a constitutively open epithelial Na⁺ channel.