Analysis of the link between the redox state and enzymatic activity of the HtrA (DegP) protein from Escherichia coli

Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically

Factors Defining the Functional Oligomeric State of Escherichia coli DegP Protease

Escherichia coli DegP protein is a periplasmic protein that functions both as a protease and as a chaperone. In the absence of substrate, DegP oligomerizes as a hexameric cage but in its presence DegP reorganizes into 12 and 24-mer cages with large chambers that house the substrate for degradation or refolding. Here, we studied the factors that determine the oligomeric state adopted by DegP in the presence of substrate. Using size exclusion chromatography and electron microscopy, we found that the size of the substrate molecule is the main factor conditioning the oligomeric state adopted by the enzyme. Other factors such as temperature, a major regulatory factor of the activity of this enzyme, did not influence the oligomeric state adopted by DegP. In addition, we observed that substrate concentration exerted an effect only when large substrates (full-length proteins) were used. However, small substrate molecules (peptides) always triggered the same oligomeric state regardless of their concentration. These results clarify important aspects of the regulation of the oligomeric state of DegP

Magnetic treatment of industrial water. Silica activation

The paper presents two large-scale observations of magnetic treatment of industrial water, aimed at investigating changes in the formation of deposits. First, a four-month experiment is described with two identical 25 kW heat exchangers, where in one case the inlet water was treated by a magneto-hydrodynamic method. Deposits recovered from both exchangers were analyzed chemically, by X-ray diffraction, infrared spectroscopy and PIXE. The amount of deposit for untreated water, composed mostly of calcite, increased exponentially with temperature reaching 20 g/m of tube at the warm end of the heat exchanger. The mass of the deposit for magnetically treated water did not depend on temperature and was only ca. 0.5 g/m of tube. It was composed of mainly noncrystalline silica-rich material. Further results were obtained from the practical installation at three blocks of a 1 GW power plant. The soft, amorphous deposit for magnetically treated water had a specific surface area of 80 m2/g and an infrared spectrum similar to that of a silicate hydrogel. Therefore, it appeared that, as a result of the passage through the magnetic device, crystallization of carbonates in water was blocked due to initiation of another, competitive process. This process is the activation of the colloidal silica, which will adsorb calcium, magnesium or other metal ions and then precipitate from the solution as the coagulated agglomerate. The most probable mechanism responsible for silica activation is a Lorentz-force induced deformation of the diffuse layer leading to the increased counterion concentration in the adsorption layer of the negatively charged silica

Economic Development as a Tool to Reduce Secessionism in Jammu and Kashmir

Conflict and instability in Jammu and Kashmir have been a major hindrance to the Indian state?s development and progress. The current lull in violence presents an opportunity to reduce secessionist sentiment and instability in the state. This report surveys the economic development potential of the most promising sectors for state development and analyzes the likelihood that their growth will reduce secessionism

The active site residue V266 of Chlamydial HtrA is critical for substrate binding during both in vitro and in vivo conditions

HtrA is a complex, multimeric chaperone and serine protease important for the virulence and survival of many bacteria. Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for severe disease pathology. C. trachomatis HtrA (CtHtrA) has been shown to be highly expressed in laboratory models of disease. In this study, molecular modelling of CtHtrA protein active site structure identified putative S1-S3 subsite residues I242, I265, and V266. These residues were altered by site-directed mutagenesis, and these changes were shown to considerably reduce protease activity on known substrates and resulted in a narrower and distinct range of substrates compared to wild type. Bacterial two-hybrid analysis revealed that CtHtrA is able to interact in vivo with a broad range of protein sequences with high affinity. Notably, however, the interaction was significantly altered in 35 out of 69 clones when residue V266 was mutated, indicating that this residue has an important function during substrate binding