Characterization of the supporting role of SecE in protein translocation

SecYEG functions as a membrane channel for protein export. SecY constitutes the protein-conducting pore, which is enwrapped by SecE in a V-shaped manner. In its minimal form SecE consists of a single transmembrane segment that is connected to a surface-exposed amphipathic α-helix via a flexible hinge. These two domains are the major sites of interaction between SecE and SecY. Specific cleavage of SecE at the hinge region, which destroys the interaction between the two SecE domains, reduced translocation. When SecE and SecY were disulfide bonded at the two sites of interaction, protein translocation was not affected. This suggests that the SecY and SecE interactions are static, while the hinge region provides flexibility to allow the SecY pore to open.     

Elucidating the Native Architecture of the YidC: Ribosome Complex

Membrane protein biogenesis in bacteria occurs via dedicated molecular systems SecYEG and YidC that function independently and in cooperation. YidC belongs to the universally conserved Oxa1/Alb3/YidC family of membrane insertases and is believed to associate with translating ribosomes at the membrane surface. Here, we have examined the architecture of the YidC:ribosome complex formed upon YidC-mediated membrane protein insertion. Fluorescence correlation spectroscopy was employed to investigate the complex assembly under physiological conditions. A slightly acidic environment stimulates binding of detergent-solubilized YidC to ribosomes due to electrostatic interactions, while YidC acquires specificity for translating ribosomes at pH-neutral conditions. The nanodisc reconstitution of the YidC to embed it into a native phospholipid membrane environment strongly enhances the YidC:ribosome complex formation. A single copy of YidC suffices for the binding of translating ribosome both in detergent and at the lipid membrane interface, thus being the minimal functional unit. Data reveal molecular details on the insertase functioning and interactions and suggest a new structural model for the YidC:ribosome complex.     

Molecular modelling as a tool for studying the disassembly of potentially leishmanicide-targeted dendrimer

Molecular modelling studies were carried out to analyse which carbonyl group is the most vulnerable to the disassembly of potentially leishmanicide-targeted dendrimer. The dendrimers were designed using myo-inositol (core and directing group), d-mannose (directing group), l-malic acid (spacer and dendron) and hydroxymethylnitrofurazone (NFOH) as a bioactive agent. The molecular models were built containing one, two and three branches. For this preliminary analysis, physicochemical properties, such as electronic density distribution and steric hindrance regarding the carbonyl groups were evaluated, and the carbon atoms in the following carbonyl groups were considered: near the core (C₁), close to the directing group (C₂), in l-malic acid (C₃) and near the bioactive agent (C₄). The most probable targeted dendrimers showed the carbonyl close to the core as the most susceptible to a nucleophilic attack, except those molecular systems containing two branches with d-mannose as the directing group, which displayed the carbonyl group near NFOH as the most likely ester breaking point.     

The Src module: an ancient scaffold in the evolution of cytoplasmic tyrosine kinases

Tyrosine kinases were first discovered as the protein products of viral oncogenes. We now know that this large family of metazoan enzymes includes nearly one hundred structurally diverse members. Tyrosine kinases are broadly classified into two groups: the transmembrane receptor tyrosine kinases, which sense extracellular stimuli, and the cytoplasmic tyrosine kinases, which contain modular ligand-binding domains and propagate intracellular signals. Several families of cytoplasmic tyrosine kinases have in common a core architecture, the “Src module,” composed of a Src-homology 3 (SH3) domain, a Src-homology 2 (SH2) domain, and a kinase domain. Each of these families is defined by additional elaborations on this core architecture. Structural, functional, and evolutionary studies have revealed a unifying set of principles underlying the activity and regulation of tyrosine kinases built on the Src module. The discovery of these conserved properties has shaped our knowledge of the workings of protein kinases in general, and it has had important implications for our understanding of kinase dysregulation in disease and the development of effective kinase-targeted therapies.     

High thermal variance in naturally incubated turtle nests produces faster offspring

The effects of climate change on populations are complex and difficult to predict, and can result in mismatches between interdependent organisms or between organisms and their environment. Reptiles with temperature-dependent sex determination may be able to compensate for potential skews in offspring sex ratio caused by climate change by selecting cooler (i.e., shadier) nest sites. Although changing nest location may prevent sex ratio skews, it may also affect thermally sensitive performance traits in offspring. I tested righting, sprinting, and swimming performance in hatchling painted turtles (Chrysemys picta), produced by female turtles from five populations across the species’ geographic range, nesting in a common-garden environment. I found that speed of hatchling performance was faster in hatchlings whose mothers originated from warmer climates, and that nests with higher mean daily variation in incubation temperature produced faster hatchlings. These results suggest that the increased temperatures predicted by climate change models could result in hatchling turtles that are faster at sprinting and swimming; however, it is not yet known how these performance measures translate into fitness.     

Small montane cloud forest fragments are important for conserving tree diversity in the Ecuadorian Andes

Montane tropical cloud forests, with their complex topography, biodiversity, high numbers of endemic species, and rapid rates of clearing, are a top global conservation priority. However, species distributions at local and landscape scales in cloud forests are still poorly understood, in part because few regions have been surveyed. Empirical work has focused on species distributions along elevation gradients, but spatial variation among forests at the same elevation is less commonly investigated. In this study, the first to compare tree communities across multiple Andean cloud forests at similar elevations, we surveyed trees in five ridge‐top forest reserves at the upper end of the ‘mid‐elevation diversity bulge’ (1900–2250 masl) in the Intag Valley, a heavily deforested region in the Ecuadorian Andes. We found that tree communities were distinct in reserves located as close as 10 to 35 km apart, and that spatially closer forests were not more similar to one another. Although larger (1500 to 6880 ha), more intact forests contained significantly more tree species (108–120 species/0.1 ha) than smaller (30 to 780 ha) ones (56–87 species/0.1 ha), each reserve had unique combinations of more common species, and contained high proportions of species not found in the others. Results thus suggest that protecting multiple cloud forest patches within this narrow elevational band is essential to conserve landscape‐level tree diversity, and that even small forest reserves contribute significantly to biodiversity conservation. These findings can be applied to create management plans to conserve and restore cloud forests in the Andes and tropical montane cloud forests elsewhere.     

EVOLUTIONARY AND ECOLOGICAL DIFFERENTIATION IN THE PANTROPICAL TO WARM-TEMPERATE SEAWEED DIGENEA SIMPLEX (RHODOPHYTA)1

Genetic differentiation among geographic isolates of the pantropical to warm-temperate red alga Digenea simplex (Wulfen) C. Agardh was investigated using random amplified polymorphic DNA (RAPD) markers, crossing studies, and temperature tolerances experiments. Eleven isolates representing populations from the Caribbean, eastern Atlantic, and Indo-West Pacific were compared. RAPD analysis clearly revealed an Indo-West Pacific group, a Caribbean/Cape Verde Islands group, and a Canary Islands group. Crossing studies showed different levels of inter fertility. In most crosses between Western Australian and Atlantic isolates, no hybrid tetrasporophytes were formed. In crosses between Caribbean and Cape Verde Islands isolates, tetrasporophytes developed, but the viability of tetraspores was reduced. Full sexual compatibility was observed among Cape Verde Islands isolates and among isolates from Bonaire. Temperature tolerance studies indicate that Pacific isolates have a broader temperature survival range than Atlantic isolates, which may be correlated to local temperature extremes. Despite the reduced level of sexual compatibility between Caribbean and Cape Verde Islands isolates, their shared position in the RAPD analysis and similar temperature responses suggest trans-Atlantic dispersal in the near geological past. In addition to their discrete position in the RAPD distance analysis, the Canary Islands isolates were significantly more cold-tolerant than the other Atlantic isolates. This finding is consistent with the hypothesis that the Canary Islands were recolonized from cold-adapted eastern Mediterranean populations after the last Pleistocene glaciation.     

Combined 1H-Detected Solid-State NMR Spectroscopy and Electron Cryotomography to Study Membrane Proteins across Resolutions in Native Environments

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Stimulation of PgdA‐dependent peptidoglycan N‐deacetylation by GpsB‐PBP A1 in Listeria monocytogenes

Listeria monocytogenes and other pathogenic bacteria modify their peptidoglycan to protect it against enzymatic attack through the host innate immune system, such as the cell wall hydrolase lysozyme. During our studies on GpsB, a late cell division protein that controls activity of the bi‐functional penicillin binding protein PBP A1, we discovered that GpsB influences lysozyme resistance of L. monocytogenes as mutant strains lacking gpsB showed an increased lysozyme resistance. Deletion of pbpA1 corrected this effect, demonstrating that PBP A1 is also involved in this. Susceptibility to lysozyme mainly depends on two peptidoglycan modifying enzymes: The peptidoglycan N‐deacetylase PgdA and the peptidoglycan O‐acetyltransferase OatA. Genetic and biochemical experiments consistently demonstrated that the increased lysozyme resistance of the ΔgpsB mutant was PgdA‐dependent and OatA‐independent. Protein‐protein interaction studies supported the idea that GpsB, PBP A1 and PgdA form a complex in L. monocytogenes and identified the regions in PBP A1 and PgdA required for complex formation. These results establish a physiological connection between GpsB, PBP A1 and the peptidoglycan modifying enzyme PgdA. To our knowledge, this is the first reported link between a GpsB‐like cell division protein and factors important for escape from the host immune system.