The dynamical response of hen egg white lysozyme to the binding of a carbohydrate ligand

It has become clear that the binding of small and large ligands to proteins can invoke significant changes in side chain and main chain motion in the fast picosecond to nanosecond timescale. Recently, the use of a dynamical proxy has indicated that changes in these motions often reflect significant changes in conformational entropy. These entropic contributions are sometimes of the same order as the total entropy of binding. Thus, it is important to understand the connections amongst motion between the manifold of states accessible to the native state of proteins, the corresponding entropy, and how this impacts the overall energetics of protein function. The interaction of proteins with carbohydrate ligands is central to a range of biological functions. Here, we examine a classic carbohydrate interaction with an enzyme: the binding of wild-type hen egg white lysozyme (HEWL) to the natural, competitive inhibitor chitotriose. Using NMR relaxation experiments, backbone amide and side chain methyl axial order parameters were obtained across apo and chitotriose-bound HEWL. Upon binding, changes in the apparent amplitude of picosecond to nanosecond main chain and side chain motions are seen across the protein. Indeed, binding of chitotriose renders a large contiguous fraction of HEWL effectively completely rigid. Changes in methyl flexibility are most pronounced closest to the binding site, but average to only a small overall change in the dynamics across the protein. The corresponding change in conformational entropy is unfavorable and estimated to be a significant fraction of the total binding entropy.     

Influence of pastoral fallow on plant root growth and soil physical and chemical characteristics in a hill pasture

Pastoral fallowing over a growing season (October–May) has a profound effect on standing biomass and sward structure, and should have an impact on below ground plant growth and soil biological activities. Two field studies were conducted to compare the effects of pastoral fallow with rotational grazing on root growth and soil physical and chemical properties. Root growth and distribution was altered by pastoral fallowing and there was significantly (P < 0.01) less root biomass at 0–50 mm depth of soil in the fallowed sward than the grazed sward. Compared with the grazed treatment, pastoral fallow increased soil air permeability at 500 mm tension by 38%, saturated hydraulic conductivity by 26%, unsaturated hydraulic conductivity at 20 mm tension by 67% and soil moisture by 10–15%, and reduced soil bulk density by 11%. Fallowing had little effect on soil nutrients both at the end of fallowing, except for small reductions in K and Mineral N levels at 0–75 mm soil depth, and two to three years after fallowing.     

New perspectives on anaerobic methane oxidation

Anaerobic methane oxidation is a globally important but poorly understood process. Four lines of evidence have recently improved our understanding of this process. First, studies of recent marine sediments indicate that a consortium of methanogens and sulphate-reducing bacteria are responsible for anaerobic methane oxidation; a mechanism of 'reverse methanogenesis' was proposed, based on the principle of interspecies hydrogen transfer. Second, studies of known methanogens under low hydrogen and high methane conditions were unable to induce methane oxidation, indicating that 'reverse methanogenesis' is not a widespread process in methanogens. Third, lipid biomarker studies detected isotopically depleted archaeal and bacterial biomarkers from marine methane vents, and indicate that Archaea are the primary consumers of methane. Finally, phylogenetic studies indicate that only specific groups of Archaea and SRB are involved in methane oxidation. This review integrates results from these recent studies to constrain the responsible mechanisms.     

Proteoglycans mediate malaria sporozoite targeting to the liver

Malaria sporozoites are rapidly targeted to the liver where they pass through Kupffer cells and infect hepatocytes, their initial site of replication in the mammalian host. We show that sporozoites, as well as their major surface proteins, the CS protein and TRAP, recognize distinct cell type-specific surface proteoglycans from primary Kupffer cells, hepatocytes and stellate cells, but not from sinusoidal endothelia. Recombinant Plasmodium falciparum CS protein and TRAP bind to heparan sulphate on hepatocytes and both heparan and chondroitin sulphate proteoglycans on stellate cells. On Kupffer cells, CS protein predominantly recognizes chondroitin sulphate, whereas TRAP binding is glycosaminoglycan independent. Plasmodium berghei sporozoites attach to heparan sulphate on hepatocytes and stellate cells, whereas Kupffer cell recognition involves both chondroitin sulphate and heparan sulphate proteoglycans. CS protein also interacts with secreted proteoglycans from stellate cells, the major producers of extracellular matrix in the liver. In situ binding studies using frozen liver sections indicate that the majority of the CS protein binding sites are associated with these matrix proteoglycans. Our data suggest that sporozoites are first arrested in the sinusoid by binding to extracellular matrix proteoglycans and then recognize proteoglycans on the surface of Kupffer cells, which they use to traverse the sinusoidal cell barrier.     

Antisense inhibition of the plastidial glucose-6-phosphate/phosphate translocator in Vicia seeds shifts cellular differentiation and promotes protein storage

The glucose-6-phosphate/phosphate translocator (GPT) acts as an importer of carbon into the plastid. Despite the potential importance of GPT for storage in crop seeds, its regulatory role in biosynthetic pathways that are active during seed development is poorly understood. We have isolated GPT1 from Vicia narbonensis and studied its role in seed development using a transgenic approach based on the seed-specific legumin promoter LeB4. GPT1 is highly expressed in vegetative sink tissues, flowers and young seeds. In the embryo, localized upregulation of GPT1 at the onset of storage coincides with the onset of starch accumulation. Embryos of transgenic plants expressing antisense GPT1 showed a significant reduction (up to 55%) in the specific transport rate of glucose-6-phosphate as determined using proteoliposomes prepared from embryos. Furthermore, amyloplasts developed later and were smaller in size, while the expression of genes encoding plastid-specific translocators and proteins involved in starch biosynthesis was decreased. Metabolite analysis and stable isotope labelling demonstrated that starch biosynthesis was also reduced, although storage protein biosynthesis increased. This metabolic shift was characterized by upregulation of genes related to nitrogen uptake and protein storage, morphological variation of the protein-storing vacuoles, and a crude protein content of mature seeds of transgenics that was up to 30% higher than in wild-type. These findings provide evidence that (1) the prevailing level of GPT1 abundance/activity is rate-limiting for the synthesis of starch in developing seeds, (2) GPT1 exerts a controlling function on assimilate partitioning into storage protein, and (3) GPT1 is essential for the differentiation of embryonic plastids and seed maturation.