Identifying sequence determinants of reduction potentials of metalloproteins

The reduction potential of an electron transfer protein is one of its most important functional characteristics. Although the type of redox site and the protein fold are the major determinants of the reduction potential of a redox-active protein, its amino acid sequence may tune the reduction potential as well. Thus, homologous proteins can often be divided into different classes, with each class characterized by a biological function and a reduction potential. Site-specific mutagenesis of the sequence determinants of the differences in the reduction potential between classes should change the reduction potential of a protein in one class to that of the other class. Here, a procedure is presented that combines energetic and bioinformatic analysis of homologous proteins to identify sequence determinants that are also good candidates for site-specific mutations, using the [4Fe–4S] ferredoxins and the [4Fe–4S] high-potential iron–sulfur proteins as examples. This procedure is designed to guide site-specific mutations or more computationally expensive studies, such as molecular dynamics simulations. To make the procedure more accessible to the general scientific community, it is being implemented into CHARMMing, a Web-based portal, with a library of density functional theory results for the redox site that are used in the setting up of Poisson–Boltzmann continuum electrostatics calculations for the protein energetics.     

Expression and Functional Characterization of Membrane-Integrated Mammalian Corticotropin Releasing Factor Receptors 1 and 2 in Escherichia coli

Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors'' binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2β as well as fragments lacking the N-terminal extracellular domain, were overproduced in E. coli. The characteristics of different CRFR2β -PhoA gene fusion products expressed in bacteria were found to be in agreement with the predicted ones in the hepta-helical membrane topology model. Recombinant histidine-tagged CRFR1α and CRFR2β expression levels and bacterial subcellular localization were evaluated by cell fractionation and Western blot analysis. Protein expression parameters were assessed, including the influence of E. coli bacterial hosts, culture media and the impact of either PelB or DsbA signal peptide. In general, the large majority of receptor proteins became inserted in the bacterial membrane. Across all experimental conditions significantly more CRFR2β product was obtained in comparison to CRFR1α. Following a detergent screen analysis, bacterial membranes containing CRFR1α and CRFR2β were best solubilized with the zwitterionic detergent FC-14. Binding of different peptide ligands to CRFR1α and CRFR2β membrane fractions were similar, in part, to the complex pharmacology observed in eukaryotic cells. We suggest that our E. coli expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies.     

Feeding ecology of Lilian’s Lovebird Agapornis lilianae in Liwonde National Park,Malawi

Lilian's Lovebird Agapornis lilianae is a small, near-threatened parrot resident in mopane Colophospermum mopane woodlands. We investigated its diet and foraging behaviour in Liwonde National Park, Malawi. We expected that Lilian's Lovebirds would show little specialisation for a particular food source but generally feed on available seeds, fruits, flowers and other items as observed in other lovebirds. Lilian's Lovebirds fed on 30 different plant species. Lilian's Lovebirds were observed feeding in six habitat types in Liwonde National Park and adjacent areas during the wet season, and four in the dry season. In the wet season lovebirds (23% of observations) foraged in grassy wetland (dambo) areas the most, whilst in the dry season they foraged in grasslands with tree cover (18%) the most. In mopane woodland, foraging flock sizes differed significantly between the wet (mean = 19.8 ± 1.0 lovebirds) and dry season (mean = 33.6 ± 2.3 lovebirds). Grass seeds were lovebirds’ main food source from December to June. The nutritional analysis of preferred foods showed that grass seeds have a relatively high protein and energy content. Grass seed availability is reduced with savanna burning and so early season burning (before May–June) in areas in and outside the park is not recommended.     

Design of a rotating disk reactor to assess the colonization of biofilms by free-living amoebae under high shear rates

The present study was aimed at designing and optimizing a rotating disk reactor simulating high hydrodynamic shear rates (γ), which are representative of cooling circuits. The characteristics of the hydrodynamic conditions in the reactor and the complex approach used to engineer it are described. A 60 l tank was filled with freshwater containing free-living amoebae (FLA) and bacteria. Adhesion of the bacteria and formation of a biofilm on the stainless steel coupons were observed. FLA were able to establish in these biofilms under γ as high as 85,000 s^?1. Several physical mechanisms (convection, diffusion, sedimentation) could explain the accumulation of amoeboid cells on surfaces, but further research is required to fully understand and model the fine mechanisms governing such transport under γ similar to those encountered in the industrial environment. This technological advance may enable research into these topics.