Cotranslational Folding Increases GFP Folding Yield

Protein sequences evolved to fold in cells, including cotranslational folding of nascent polypeptide chains during their synthesis by the ribosome. The vectorial (N- to C-terminal) nature of cotranslational folding constrains the conformations of the nascent polypeptide chain in a manner not experienced by full-length chains diluted out of denaturant. We are still discovering to what extent these constraints affect later, posttranslational folding events. Here we directly address whether conformational constraints imposed by cotranslational folding affect the partitioning between productive folding to the native structure versus aggregation. We isolated polyribosomes from Escherichia coli cells expressing GFP, analyzed the nascent chain length distribution to determine the number of nascent chains that were long enough to fold to the native fluorescent structure, and calculated the folding yield for these nascent chains upon ribosome release versus the folding yield of an equivalent concentration of full-length, chemically denatured GFP polypeptide chains. We find that the yield of native fluorescent GFP is dramatically higher upon ribosome release of nascent chains versus dilution of full-length chains from denaturant. For kinetically trapped native structures such as GFP, folding correctly the first time, immediately after release from the ribosome, can lead to lifelong population of the native structure, as opposed to aggregation.     

Phenomorphology and ecomorphological characters of Maytenus senegalensis L. Shrublands in the Iberian Peninsula: A comparison with other Mediterranean plant communities

This work sets out to establish ecomorphological and phenomorphological patterns in the constituent flora of an Iberian Maytenus senegalensis plant community in order to relate it with or separate it from other types of Mediterranean vegetation. This plant community contains tall shrubs, scrubs, palms and vines of a variety of families. Typical ecomorphological adaptations to the Mediterranean climate were observed in the community studied: an abundance of thorny species or species with tomentose leaves and photosynthetic stems, small leaves and a high presence of species with organs capable of post-fire regeneration. However, the community also showed hybrid characteristics between scrubland (predominantly malacophyllous leaves lasting 9 months on average and low biomass) and tall shrubland communities (predominance of phanerophytes up to 3 m in height, predominantly smooth bark, periodical reposition of leaves, mean lifespan of plants 25 years and mainly fleshy fruit). The community also presents an outstanding quantity of amphiphytes (29%). From a phenological point of view, the formation of buds in winter, flowering in spring, summer–autumn seed dispersal and leaf shedding in summer are typical of other Mediterranean communities, while fructification and growth lasting three seasons, as in the case of Maytenus shrubland, is not. The RVA phenophasic index (balance between reproductive and vegetative phenophases) with values of 0.4–2 shows an intermediate value between typical shrubland (0.3–1) and forest community (1–2) values. The APC index (period of phenophasic activity of the community) differentiates it from others, since it remains at 100% for seven months, three of which are winter months. Phenophasic patterns (overlapping, or not, of flower bud formation, flowering and growth) show how similar M. senegalensis shrubland is to forests because of the absence of phenophase pattern type A (characteristic in open-scrublands); in addition, we have detected a new pattern (type G), still regarded as exclusive to M. senegalensis. As regards the existence of coenomorphs (group of species with similar morpho-functional strategies), the studied community showed much greater diversity than other communities studied to date, with an Strategies Richness index (SRi) of 0.79 (11 groups covering the 14 species existing), pointing to the co-existence of a great variety of eco-phenomorphological strategies, each with a successful response to the same ecological conditions. Finally, some of the species in the community, e.g. Asparagus horridus, Chamaerops humilis and M. senegalensis, stand out because of their unique ecomorphological and phenological characteristics.     

Water use efficiency of twenty-five co-existing Patagonian species growing under different soil water availability

The variation of plant water use efficiency (WUE) with water availability has two interacting components: a plastic response, evident when individuals of the same genotype are compared (e.g. wet versus dry years), and an interspecific response, evident when different species living in habitats with different water availability are compared. We analysed the WUE of 25 Patagonian species that belong to four life forms (grasses, shrubs, annual herbs and perennial herbs) in relation to the climatic conditions of 2 years and the mean historic water availability experienced by each species. To estimate water availability, we calculated the effective soil water potential (EWP) of each species, based on available information about soil water dynamics, phenology and root system structure. To estimate WUE, we used isotopic discrimination of leaf C (Δ¹³C) and mean annual water vapour difference between leaves and atmosphere (Δe) measured in situ. For the plastic response, for every species and life form, WUE increased from the dry to the wet year. We hypothesize that photosynthesis was less nutrient limited in the wet than in the dry year, facilitating higher net photosynthesis rates per unit of stomatal conductance in the wet year. For the interspecific response, WUE was lower in species native to drier habitats than in species native to wetter habitats. This response was mostly accounted for by a decrease in Δe with EWP. Annual herbs, which avoid drought in time (they have the earliest growth cycle), and shrubs, which avoid drought in space (they have the deepest roots), showed the highest EWP and WUE. We conclude that the conventional wisdom which states that the highest WUE occurs within a species during the driest years, and among species in the driest habitats, does not always hold true, and that co-existing life forms drastically differ in water availability and water economy.     

The N-terminal region of the starch-branching enzyme from Phaseolus vulgaris L. is essential for optimal catalysis and structural stability

Starch-branching enzymes (SBEs) play a pivotal role in determining the fine structure of starch by catalyzing the syntheses of alpha-1,6-branch points. They are the members of the alpha-amylase family and have four conserved regions in a central (beta/alpha)8 barrel, including the catalytic sites. Although the role of the catalytic barrel domain of an SBE is known, that of its N- and C-terminal regions remain unclear. We have previously shown that the C-terminal regions of the two SBE isozymes (designated as PvSBE1 and PvSBE2) from kidney bean (Phaseolus vulgaris L.) have different roles in branching enzyme activity. To understand the contribution of the N-terminal region to catalysis, six chimeric enzymes were constructed between PvSBE1 and PvSBE2. Only one enzyme (1Na/2Nb)-II, in which a portion of the N-terminal region of PvSBE2 was substituted by the corresponding region of PvSBE1, retained 6% of the PvSBE2 activity. The N-terminal truncated form (DeltaN46-PvSBE2), lacking 46 N-terminal residues of PvSBE2, lost enzyme activity and stability to proteolysis. To investigate the possible function of this region, three residues (Asp-15, His-24, and Arg-28) among these 46 residues were subjected to site-directed mutagenesis. The purified mutant enzymes showed nearly the same K(m) values as PvSBE2 but had lower V(max) values and heat stabilities than PvSBE2. These results suggest that the N-terminal region of the kidney bean SBE is essential for maximum enzyme activity and thermostability.     

Thematic review series: Adipocyte Biology. Adipocyte stress: the endoplasmic reticulum and metabolic disease

In the context of obesity and its related maladies, the adipocyte plays a central role in the balance, or imbalance, of metabolic homeostasis. An obese, hypertrophic adipocyte is challenged by many insults, including surplus energy, inflammation, insulin resistance, and considerable stress to various organelles. The endoplasmic reticulum (ER) is one such vital organelle that demonstrates significant signs of stress and dysfunction in obesity and insulin resistance. Under normal conditions, the ER must function in the unique and trying environment of the adipocyte, adapting to meet the demands of increased protein synthesis and secretion, energy storage in the form of triglyceride droplet formation, and nutrient sensing that are particular to the differentiated fat cell. When nutrients are in pathological excess, the ER is overwhelmed and the unfolded protein response (UPR) is activated. Remarkably, the consequences of UPR activation have been causally linked to the development of insulin resistance through a multitude of possible mechanisms, including c-jun N-terminal kinase activation, inflammation, and oxidative stress. This review will focus on the function of the ER under normal conditions in the adipocyte and the pathological effects of a stressed ER contributing to adipocyte dysfunction and a thwarted metabolic homeostasis.     

BPPred: a Web-based computational tool for predicting biophysical parameters of proteins

We exploit the availability of recent experimental data on a variety of proteins to develop a Web-based prediction algorithm (BPPred) to calculate several biophysical parameters commonly used to describe the folding process. These parameters include the equilibrium m-values, the length of proteins, and the changes upon unfolding in the solvent-accessible surface area, in the heat capacity, and in the radius of gyration. We also show that the knowledge of any one of these quantities allows an estimate of the others to be obtained, and describe the confidence limits with which these estimations can be made. Furthermore, we discuss how the kinetic m-values, or the Beta Tanford values, may provide an estimate of the solvent-accessible surface area and the radius of gyration of the transition state for protein folding. Taken together, these results suggest that BPPred should represent a valuable tool for interpreting experimental measurements, as well as the results of molecular dynamics simulations.     

Insertion polymorphism of transposable elements and population structure of Anopheles gambiae M and S molecular forms in Cameroon

The insertion polymorphism of five transposable element (TE) families was studied by Southern blots in several populations of the M and S molecular forms of the mosquito Anopheles gambiae sensu stricto from southern Cameroon. We showed that the mean TE insertion site number and the within-population insertion site polymorphism globally differed between the M and S molecular forms. The comparison of the TE insertion profiles of the populations revealed a significant differentiation between these two molecular forms (0.163 < Phi(ST) < 0.371). We cloned several insertions of a non-LTR retrotransposon (Aara8) that were fixed in one form and absent in the other one. The only insertion that could be clearly located on a chromosome arm mapped to cytological division 6 of chromosome X, confirming the importance of this region in the ongoing speciation between the M and S molecular forms.     

Pleistocene refugia in an arid landscape: analysis of a widely distributed Australian passerine

While many studies have documented the effect that glacial cycles have had on northern hemisphere species, few have attempted to study the associated effect of aridification at low latitudes in the southern hemisphere. We investigated the past effects that cyclic aridification may have had on the population structure and history of a widespread endemic Australian bird species, the Australian magpie (Gymnorhina tibicen). One thousand one hundred and sixty-six samples from across its native range were analysed for mitochondrial control region sequence variation and variation at six microsatellite loci. Analysis of mitochondrial control region sequence data indicated monophyletic clades that were geographically congruent with an eastern and western region. The contemporary distribution of east and west clades is nonoverlapping but in close proximity. Populations were estimated to have diverged in the Pleistocene around 36,000 years ago. The putative Carpentarian and Nullarbor arid barriers appear to be associated with the divergence between east and west mainland populations. Nested clade analysis indicated a signature of range expansion in the eastern region suggesting movement possibly inland and northward subsequent to the last period of aridity. The island population of Tasmania was of very recent origin, possibly since sea levels rose 16,000 years ago. Given the east-west structure, there was no congruence between morphology and recent history of this species indicating a lack of support for morphological taxa. Overall mitochondrial DNA and microsatellite variation suggest that increasing aridity and Pleistocene refugia played a role in structuring populations of the Australian magpie; however, the dispersal ability and generalist habitat requirements may have facilitated the movement of magpies into an almost contiguous modern distribution across the continent. This study supports the idea that Pleistocene aridification played an important role in structuring intraspecific variation in low latitudinal southern hemisphere avian species.     

Diversity in forms and functions of carbonic anhydrase in terrestrial higher plants

The review summarizes current data on the existence in terrestrial higher plants of several carbonic anhydrase forms differing in their properties, molecular structure, and intracellular localization. Possible functions of these carbonic anhydrases are discussed as well as specific features of carbon-concentrating mechanisms in phototrophic tissues of plants with C₃ and C₄ pathways of photosynthesis.