A c Subunit with Four Transmembrane Helices and One Ion (Na+)-binding Site in an Archaeal ATP Synthase: IMPLICATIONS FOR c RING FUNCTION AND STRUCTURE*

The ion-driven membrane rotors of ATP synthases consist of multiple copies of subunit c, forming a closed ring. Subunit c typically comprises two transmembrane helices, and the c ring features an ion-binding site in between each pair of adjacent subunits. Here, we use experimental and computational methods to study the structure and specificity of an archaeal c subunit more akin to those of V-type ATPases, namely that from Pyrococcus furiosus. The c subunit was purified by chloroform/methanol extraction and determined to be 15.8 kDa with four predicted transmembrane helices. However, labeling with DCCD as well as Na⁺-DCCD competition experiments revealed only one binding site for DCCD and Na⁺, indicating that the mature c subunit of this A₁A_O ATP synthase is indeed of the V-type. A structural model generated computationally revealed one Na⁺-binding site within each of the c subunits, mediated by a conserved glutamate side chain alongside other coordinating groups. An intriguing second glutamate located in-between adjacent c subunits was ruled out as a functional Na⁺-binding site. Molecular dynamics simulations indicate that the c ring of P. furiosus is highly Na⁺-specific under in vivo conditions, comparable with the Na⁺-dependent V₁V_O ATPase from Enterococcus hirae. Interestingly, the same holds true for the c ring from the methanogenic archaeon Methanobrevibacter ruminantium, whose c subunits also feature a V-type architecture but carry two Na⁺-binding sites instead. These findings are discussed in light of their physiological relevance and with respect to the mode of ion coupling in A₁A_O ATP synthases.     

An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography

Background  

Functional Genomics, the systematic characterisation of the functions of an organism's genes, includes the study of the gene products, the proteins. Such studies require methods to express and purify these proteins in a parallel, time and cost effective manner.     

Structuring heterogeneous biological information using fuzzy clustering of k-partite graphs

Background  

Extensive and automated data integration in bioinformatics facilitates the construction of large, complex biological networks. However, the challenge lies in the interpretation of these networks. While most research focuses on the unipartite or bipartite case, we address the more general but common situation of k-partite graphs. These graphs contain k different node types and links are only allowed between nodes of different types. In order to reveal their structural organization and describe the contained information in a more coarse-grained fashion, we ask how to detect clusters within each node type.     

Organic Carbon Degradation in Anoxic Organic-Rich Shelf Sediments: Biogeochemical Rates and Microbial Abundance

Identifying and explaining bottlenecks in organic carbon mineralization and the persistence of organic matter in marine sediments remain challenging. This study aims to illuminate the process of carbon flow between microorganisms involved in the sedimentary microbial food chain in anoxic, organic-rich sediments of the central Namibian upwelling system, using biogeochemical rate measurements and abundances of Bacteroidetes, Gammaproteobacteria, and sulfate-reducing bacteria at two sampling stations. Sulfate reduction rates decreased by three orders of magnitude in the top 20 cm at one sampling station (280 nmol cm^?3 d^?1 – 0.1 nmol cm^?3 d^?1) and by a factor of 7 at the second station (65 nmol cm^?3 d^?1 – 9.6 nmol cm^?3 d^?1). However, rates of enzymatic hydrolysis decreased by less than a factor of three at both sampling stations for the polysaccharides laminarin (23 nmol cm^?3 d^?1– 8 nmol cm^?3 d^?1 and 22 nmol cm^?3 d^?1– 10 nmol cm^?3 d^?1) and pullulan (11 nmol cm^?3 d^?1– 4 nmol cm^?3 d^?1 and 8 nmol cm^?3 d^?1– 6 nmol cm^?3 d^?1). Increasing imbalance between carbon turnover by hydrolysis and terminal oxidation with depth, the steep decrease in cell specific activity of sulfate reducing bacteria with depth, low concentrations of volatile fatty acids (less than 15 μM), and persistence of dissolved organic carbon, suggest decreasing bioavailability and substrate limitation with depth.     

Induced circular dichroism of the interaction between pinacyanol and algal alginates

The interaction between pinacyanol chloride and sodium alginate or guluronate-rich alginate is found to effect profound changes in the visible absorbance and circular dichroism spectra. Two different types of aggregates are observed depending on the relative dye/alginate concentrations. With a dye/alginate ratio at 1:1, a complex is deduced based on an analysis of Job’s method and conductometric titrations. Another complex forms at 1:10 dye/alginate ratio and only in the presence of alginate or guluronate-rich alginate. The two aggregates are in dynamic equilibrium according to the presence of isosbestic points in the visible spectra. The effects of pH and divalent cations on the spectra are studied. The 1:10 complex is damaged by addition of hydrochloric acid and divalent cations; however, at low concentration of these agents the spectra indicate conversion of the complex into the 1:1 aggregate. Models for the two complexes are proposed taking into account the preference of guluronate binding sites for chelating ions.     

Individual variability and population regulation: a model of the significance of within-generation density dependence

Most models of theoretical population ecology consider population density as a state variable and thus ignore the fact that populations are composed not of identical average individuals but of individuals which are usually different. However, this individual variability may be important for population regulation. We therefore analysed an individual-based population model which explicitly describes within-generation processes, i.e. individual growth, starvation, and resource dynamics. The results show that if population dynamics are dominated by slow changes in resource level, the population size in the model undergoes wide oscillation, often leading to extinction. If, on the other hand, fast within-generation processes predominate, such as starvation and sudden drops in resource levels, the population fluctuates to a limited extent around an average. Within-generation density dependence may thus be an important mechanism which is largely ignored in classic time-discrete state-variable models. We conclude that the individual-based approach provides important insights into the hierarchical organization of population dynamics, i.e. the relationship between fast processes at the individual level and slower processes at the population level.     

Binding of bovine parathyroid hormone to surface receptors of cultured B-lymphocytes

Binding of parathyroid hormone onto B-lymphocytes is detected by the utilization of the labelled antibody membrane assay. The amount of parathyroid hormone bound to the receptor sites was depending on the quantity of cells in the incubation milieu. Each cell line showed typical characteristics in time course of parathyroid hormone binding and maximal receptor capacity. Fragmentation of intact parathyroid hormone, also varying with the cell line tested, was very rapid, even at 24°C. Within 20 min most of the cell lines destroyed 50% of the native hormone in the incubation mixture, indicating a fragmentation rate of up to 2.25 ng/min at 37°C. B_max and K_D for the different lymphocytes was 5.3–19 · 10¹¹ M and 1.8–18.5 · 10¹¹ M, respectively. These values are in the range of reported plasma concentrations and may therefore represent more physiological values for the capacity and affinity of membrane receptors.