Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gα_i. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.     

Mice Lacking Platelet-Derived Growth Factor D Display a Mild Vascular Phenotype

Platelet-derived growth factor D (PDGF-D) is the most recently discovered member of the PDGF family. PDGF-D signals through PDGF receptor β, but its biological role remains largely unknown. In contrast to other members of the PDGF family of growth factors, which have been extensively investigated using different knockout approaches in mice, PDGF-D has until now not been characterized by gene inactivation in mice. Here, we present the phenotype of a constitutive Pdgfd knockout mouse model (Pdgfd^-/-), carrying a LacZ reporter used to visualize Pdgfd promoter activity. Inactivation of the Pdgfd gene resulted in a mild phenotype in C57BL/6 mice, and the offspring was viable, fertile and generally in good health. We show that Pdgfd reporter gene activity was consistently localized to vascular structures in both postnatal and adult tissues. The expression was predominantly arterial, often localizing to vascular bifurcations. Endothelial cells appeared to be the dominating source for Pdgfd, but reporter gene activity was occasionally also found in subpopulations of mural cells. Tissue-specific analyses of vascular structures revealed that NG2-expressing pericytes of the cardiac vasculature were disorganized in Pdgfd^-/- mice. Furthermore, Pdgfd^-/- mice also had a slightly elevated blood pressure. In summary, the vascular expression pattern together with morphological changes in NG2-expressing cells, and the increase in blood pressure, support a function for PDGF-D in regulating systemic arterial blood pressure, and suggests a role in maintaining vascular homeostasis.     

Diffusion Retardation by Binding of Tobramycin in an Alginate Biofilm Model

Microbial cells embedded in a self-produced extracellular biofilm matrix cause chronic infections, e. g. by Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. The antibiotic killing of bacteria in biofilms is generally known to be reduced by 100–1000 times relative to planktonic bacteria. This makes such infections difficult to treat. We have therefore proposed that biofilms can be regarded as an independent compartment with distinct pharmacokinetics. To elucidate this pharmacokinetics we have measured the penetration of the tobramycin into seaweed alginate beads which serve as a model of the extracellular polysaccharide matrix in P. aeruginosa biofilm. We find that, rather than a normal first order saturation curve, the concentration of tobramycin in the alginate beads follows a power-law as a function of the external concentration. Further, the tobramycin is observed to be uniformly distributed throughout the volume of the alginate bead. The power-law appears to be a consequence of binding to a multitude of different binding sites. In a diffusion model these results are shown to produce pronounced retardation of the penetration of tobramycin into the biofilm. This filtering of the free tobramycin concentration inside biofilm beads is expected to aid in augmenting the survival probability of bacteria residing in the biofilm.     

Using quantitative redox proteomics to dissect the yeast redoxome

To understand and eventually predict the effects of changing redox conditions and oxidant levels on the physiology of an organism, it is essential to gain knowledge about its redoxome: the proteins whose activities are controlled by the oxidation status of their cysteine thiols. Here, we applied the quantitative redox proteomic method OxICAT to Saccharomyces cerevisiae and determined the in vivo thiol oxidation status of almost 300 different yeast proteins distributed among various cellular compartments. We found that a substantial number of cytosolic and mitochondrial proteins are partially oxidized during exponential growth. Our results suggest that prevailing redox conditions constantly control central cellular pathways by fine-tuning oxidation status and hence activity of these proteins. Treatment with sublethal H(2)O(2) concentrations caused a subset of 41 proteins to undergo substantial thiol modifications, thereby affecting a variety of different cellular pathways, many of which are directly or indirectly involved in increasing oxidative stress resistance. Classification of the identified protein thiols according to their steady-state oxidation levels and sensitivity to peroxide treatment revealed that redox sensitivity of protein thiols does not predict peroxide sensitivity. Our studies provide experimental evidence that the ability of protein thiols to react to changing peroxide levels is likely governed by both thermodynamic and kinetic parameters, making predicting thiol modifications challenging and de novo identification of peroxide sensitive protein thiols indispensable.     

An ancient look at UCP1

Brown adipose tissue serves as a thermogenic organ in placental mammals to defend body temperature in the cold by nonshivering thermogenesis. The thermogenic function of brown adipose tissue is enabled by several specialised features on the organ as well as on the cellular level, including dense sympathetic innervation and vascularisation, high lipolytic capacity and mitochondrial density and the unique expression of uncoupling protein 1 (UCP1). This mitochondrial carrier protein is inserted into the inner mitochondrial membrane and stimulates maximum mitochondrial respiration by dissipating proton-motive force as heat. Studies in knockout mice have clearly demonstrated that UCP1 is essential for nonshivering thermogenesis in brown adipose tissue. For a long time it had been presumed that brown adipose tissue and UCP1 emerged in placental mammals providing them with a unique advantage to survive in the cold. Our subsequent discoveries of UCP1 orthologues in ectotherm vertebrates and marsupials clearly refute this presumption. We can now initiate comparative studies on the structure-function relationships in UCP1 orthologues from different vertebrates to elucidate when during vertebrate evolution UCP1 gained the biochemical properties required for nonshivering thermogenesis.     

Ethanol production from hexoses, pentoses, and dilute-acid hydrolyzate by Mucor indicus

Consumption of hexoses and pentoses and production of ethanol by Mucor indicus were investigated in both synthetic media and dilute-acid hydrolyzates. The fungus was able to grow in a poor medium containing only carbon, nitrogen, phosphate, potassium, and magnesium sources. However, the cultivation took more than a week and the ethanol yield was only 0.2 gg(-1). Enrichment of the medium by addition of trace metals, particularly zinc and yeast extract, improved the growth rate and yield, such that the cultivation was completed in less than 24 h and the ethanol and biomass yields were increased to 0.40 and 0.20 gg(-1), respectively. The fungus was able to assimilate glucose, galactose, mannose, and xylose, and produced ethanol with yields of 0.40, 0.34, 0.39, and 0.18 gg(-1), respectively. However, arabinose was poorly consumed and no formation of ethanol was detected. Glycerol was the major by-product in the cultivation on the hexoses, while formation of glycerol and xylitol were detected in the cultivation of the fungus on xylose. The fungus was able to take up the sugars present in dilute-acid hydrolyzate as well as the inhibitors, acetic acid, furfural, and hydroxymethyl furfural. M. indicus was able to grow under anaerobic conditions when glucose was the sole carbon source, but not on xylose or the hydrolyzate. The yield of ethanol in anaerobic cultivation on glucose was 0.46 g g(-1).     

Cryo-electron microscopy of vitreous sections

Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction.     

Origin and evolution of the northern hemisphere disjunction in the moss genus Homalothecium (Brachytheciaceae)

Competing hypotheses that rely either on a stepping-stone dispersal via the North Atlantic or the Bering land bridges, or more recent transoceanic dispersal, have been proposed to explain the disjunct distribution of Mediterranean flora in southern Europe and western North America. These hypotheses were tested with molecular dating using a phylogeny of the moss genus Homalothecium based on ITS, atpB-rbcL, and rpl16 sequence data. The monophyly of two main lineages in Western Palearctic (Europe, central Asia and north Africa) and North America is consistent with the ancient vicariance hypothesis. The monophyly of Madeiran H. sericeum accessions supports the recognition of the Macaronesian endemic H. mandonii. A range of absolute rates of molecular evolution documented in land plants was used as probabilistic calibration prior by a Bayesian inference implementing a relaxed-clock model to derive ages for the nodes of interest. Our age estimates for the divergence of the American and Western Palearctic Homalothecium clade (5.7 Ma, IC 3.52-8.26) and the origin of H. mandonii (2.52 Myr IC 0.86-8.25) are not compatible with the ancient vicariance hypothesis. Age estimates suggests that species distributions result from rare instances of dispersal and subsequent sympatric diversification. The calibrated phylogeny indicates that Homalothecium has undergone a fast radiation during the last 4 Myr, which is consistent with the low levels of morphological divergence among sibling species.