An error analysis for two-state protein-folding kinetic parameters and phi-values: progress toward precision by exploring pH dependencies on Leffler plots

The interpretation of φ-values has led to an understanding of the folding transition state ensemble of a variety of proteins. Although the main guidelines and equations for calculating φ are well established, there remains some controversy about the quality of the numerical values obtained. By analyzing a complete set of results from kinetic experiments with the SH3 domain of α-spectrin (Spc-SH3) and applying classical error methods and error-propagation formulas, we evaluated the uncertainties involved in two-state-folding kinetic experimental parameters and the corresponding calculated φ-values. We show that kinetic constants in water and m values can be properly estimated from a judicious weighting of fitting errors and describe some procedures to calculate the errors in Gibbs energies and φ-values from a traditional two-point Leffler analysis. Furthermore, on the basis of general assumptions made with the protein engineering method, we show how to generate multipoint Leffler plots via the analysis of pH dependencies of kinetic parameters. We calculated the definitive φ-values for a collection of single mutations previously designed to characterize the folding transition state of the α-spectrin SH3 domain. The effectiveness of the pH-scanning procedure is also discussed in the context of error analysis. Judging from the magnitudes of the error bars obtained from two-point and multipoint Leffler plots, we conclude that the precision obtained for φ-values should be ∼25%, a reasonable limit that takes into account the propagation of experimental errors.     

Unraveling the mechanisms of tryptophan fluorescence quenching in the triosephosphate isomerase from Giardia lamblia

In the native state several proteins exhibit a quenching of fluorescence of their tryptophans. We studied triosephosphate isomerase from Giardia lamblia (GlTIM) to dissect the mechanisms that account for the quenching of fluorescence of its Trp. GlTIM contains four Trp per monomer (Trp75, Trp162, Trp173, and Trp196) distributed throughout the 3D structure. The fluorescence of the denatured enzyme is 3-fold higher than that of native GlTIM. To ascertain the origin of this phenomenon, single and triple mutants of Trp per Phe were made. The intrinsic fluorescence was determined, and the data were interpreted on the basis of the crystal structure of the enzyme. Our data show that the fluorescence of all Trp residues is quenched through two different mechanisms. In one, fluorescence is quenched by aromatic-aromatic interactions due to the proximity and orientation of the indole groups of Trp196 and Trp162. The magnitude of the quenching of fluorescence in Trp162 is higher than in the other three Trp. Fluorescence quenching is also due to energy transfer to the charged residues that surround Trp 75, 173 and 196. Further analysis of the fluorescence of GlTIM showed that, among TIMs from other parasites, Trp at position 12 exhibits rather unique properties.     

Comparative effect of the fungicide Prochloraz-Mn on Agaricus bisporus vegetative-mycelium and fruit-body cell walls.

Fungicides to control mycopathogens of commercial Agaricus bisporus, a mushroom cultivated for human consumption, are a major field of study, since these chemicals are toxic to both the host and its fungal parasites. The fungicide Prochloraz-Mn, used at its LD50 for A. bisporus, partially inhibited protein biosynthesis in the vegetative mycelial cell walls of this mushroom and caused significant changes in cell-wall polysaccharide structure, as deduced by methylation analysis and gas liquid chromatography-mass spectrometry (GLC-MS). Furthermore, the aggregated mycelial walls showed distinct alterations in their overall chemical composition following the administration of Prochloraz-Mn at the LD50 and the LD50 x1000. As expected, GLC-MS studies indicated that the latter dose caused more appreciable differences in polysaccharide structure. The decrease in mushroom crop yields obtained from industrial cultures treated with Prochloraz-Mn to control V. fungicola infection depended on the dose of the fungicide employed, whereas fruit-body morphology was only slightly affected at the highest Prochloraz-Mn concentration used.     

p32 (gC1qBP) is a general protein kinase C (PKC)-binding protein; interaction and cellular localization of P32-PKC complexes in ray hepatocytes.

The aim of this study was to identify cellular proteins that bind protein kinase C (PKC) and may influence its activity and its localization. A 32-kDa PKC-binding protein was purified to homogeneity from the Triton X-100-insoluble fraction obtained from hepatocytes homogenates. The protein was identified by NH(2)-terminal amino acid sequencing as the previously described mature form of p32 (gC1qR). Recombinant p32 was expressed as a glutathione S-transferase fusion protein, affinity-purified, and tested for an in vitro interaction with PKC using an overlay assay approach. All PKC isoforms expressed in rat hepatocytes interacted in vitro with p32, but the binding dependence on PKC activators was different for each one. Whereas PKCdelta only binds to p32 in the presence of PKC activators, PKCzeta and PKCalpha increase their binding when they are in the activated form. Other PKC isoforms such as beta, epsilon, and theta bind equally well to p32 regardless of the presence of PKC activators, and PKCmu binds even better in their absence. It was also found that p32 is not a substrate for any of the PKC isoforms tested, but interestingly, its presence had a stimulatory effect (2-fold for PKCdelta) on PKC activity. We also observed in vivo interaction between PKC and p32 by immunofluorescence and confocal microscopy. A time course of phorbol ester treatment of cultured rat hepatocytes (C9 cells) showed that PKCtheta and p32 are constitutively associated in vivo, whereas PKCdelta activation is required for its association with p32. Our data also showed that phorbol ester treatment induces a transient translocation of p32 from the cytoplasm to the cell nucleus. Together, these findings suggest that p32 may be a regulator of PKC location and function.     

Pathogen-derived resistance to Citrus tristeza virus (CTV) in transgenic mexican lime (Citrus aurantifolia (Christ.) Swing.) plants expressing its p25 coat protein gene

The p25 coat protein (CP) gene of Citrustristezavirus (CTV) was incorporated to Mexican lime plants and forty-twotransgeniclines were produced, 25 containing the p25 CP gene of thesevere CTV strain T-305 and 17 with that of the mild strain T-317. When plantspropagated from each transgenic line were graft-inoculated with CTV T-305 oraphidinoculated with T-300, two types of response to viral challenge wereobserved: some lines developed CTV symptoms similar to those of non-transgeniccontrols, whereas others exhibited protection against the virus. Thisprotectionconsisted of a proportion of plants, ranging from 10 to 33%, that wereresistantto CTV, and the rest of them that showed a significant delay in virusaccumulation and symptom onset. Protection was efficient against non-homologousCTV strains and was generally accompanied by high accumulation of p25 CP in theprotected lines, which suggest a CP-mediated protection mechanism in mostcases.This is the first report demonstrating pathogen-derived resistance intransgenicplants against a Closterovirus member in its natural host.     

Metagenomic Analysis from the Interior of a Speleothem in Tjuv-Ante's Cave,Northern Sweden

Speleothems are secondary mineral deposits normally formed by water supersaturated with calcium carbonate percolating into underground caves, and are often associated with low-nutrient and mostly non-phototrophic conditions. Tjuv-Ante’s cave is a shallow-depth cave formed by the action of waves, with granite and dolerite as major components, and opal-A and calcite as part of the speleothems, making it a rare kind of cave. We generated two DNA shotgun sequencing metagenomic datasets from the interior of a speleothem from Tjuv-Ante’s cave representing areas of old and relatively recent speleothem formation. We used these datasets to perform i) an evaluation of the use of these speleothems as past biodiversity archives, ii) functional and taxonomic profiling of the speleothem’s different formation periods, and iii) taxonomic comparison of the metagenomic results to previous microscopic analyses from a nearby speleothem of the same cave. Our analyses confirm the abundance of Actinobacteria and fungi as previously reported by microscopic analyses on this cave, however we also discovered a larger biodiversity. Interestingly, we identified photosynthetic genes, as well as genes related to iron and sulphur metabolism, suggesting the presence of chemoautotrophs. Furthermore, we identified taxa and functions related to biomineralization. However, we could not confidently establish the use of this type of speleothems as biological paleoarchives due to the potential leaching from the outside of the cave and the DNA damage that we propose has been caused by the fungal chemical etching.     

When the Background Matters: Using Scenarios from Integrated Assessment Models in Prospective Life Cycle Assessment

Prospective life cycle assessment (LCA) needs to deal with the large epistemological uncertainty about the future to support more robust future environmental impact assessments of technologies. This study proposes a novel approach that systematically changes the background processes in a prospective LCA based on scenarios of an integrated assessment model (IAM), the IMAGE model. Consistent worldwide scenarios from IMAGE are evaluated in the life cycle inventory using ecoinvent v3.3. To test the approach, only the electricity sector was changed in a prospective LCA of an internal combustion engine vehicle (ICEV) and an electric vehicle (EV) using six baseline and mitigation climate scenarios until 2050. This case study shows that changes in the electricity background can be very important for the environmental impacts of EV. Also, the approach demonstrates that the relative environmental performance of EV and ICEV over time is more complex and multifaceted than previously assumed. Uncertainty due to future developments manifests in different impacts depending on the product (EV or ICEV), the impact category, and the scenario and year considered. More robust prospective LCAs can be achieved, particularly for emerging technologies, by expanding this approach to other economic sectors beyond electricity background changes and mobility applications as well as by including uncertainty and changes in foreground parameters. A more systematic and structured composition of future inventory databases driven by IAM scenarios helps to acknowledge epistemological uncertainty and to increase the temporal consistency of foreground and background systems in LCAs of emerging technologies.     

Differentiation and wall chemistry of Agaricus bisporus vegetative and aggregated micelia

Changes in the chemical composition of isolated cell walls and fractions were encountered during the differentiation of vegetative and aggregated mycelia of Agaricus bisporus.Differentiation was accompanied by quantitative variations of the wall polysaccharidic components. Neutral carbohydrates were composed of glucose, galactose, mannose and xylose and glucosamine as the only amino sugar. Differences in wall chemistry were correlated to the secondary and tertiary mycelial forms.     

Species differences in adrenocortical activity of new world primates: Response to dexamethasone suppression

The effect of dexamethasone-induced alteration of adrenocortical activity was assessed in two species of New World primates: Saimiri sciureus and Callicebus moloch. Basal and stress levels of corticosteroids were determined following treatment with three doses of dexamethasone (0, 50, or 500 μg/kg). Saimiri showed substantially higher basal levels of corticosteroids than did Callicebus and a greater incremental corticosteroid response to physical restraint. Dexamethasone was found to reduce basal corticosteroids in both species, although this effect was greater for Callicebus than for Saimiri Moreover, the adrenocortical response to physical restraint was reduced by dexamethasone treatment in Callicebus but not in Saimiri. The pattern of results suggests that the species differ in the mechanisms mediating adrenocortical activity.