Functional Dynamics of Hexameric Helicase Probed by Hydrogen Exchange and Simulation

The biological function of large macromolecular assemblies depends on their structure and their dynamics over a broad range of timescales; for this reason, it is a significant challenge to investigate these assemblies using conventional experimental techniques. One of the most promising experimental techniques is hydrogen-deuterium exchange detected by mass spectrometry. Here, we describe to our knowledge a new computational method for quantitative interpretation of deuterium exchange kinetics and apply it to a hexameric viral helicase P4 that unwinds and translocates RNA into a virus capsid at the expense of ATP hydrolysis. Room-temperature dynamics probed by a hundred nanoseconds of all-atom molecular dynamics simulations is sufficient to predict the exchange kinetics of most sequence fragments and provide a residue-level interpretation of the low-resolution experimental results. The strategy presented here is also a valuable tool to validate experimental data, e.g., assignments, and to probe mechanisms that cannot be observed by x-ray crystallography, or that occur over timescales longer than those that can be realistically simulated, such as the opening of the hexameric ring.     

Preferring thin people does not imply derogating fat people. A rasch analysis of the implicit weight attitude

Objective:

The meaning of the implicit weight attitude in individuals of different weight by distinguishing the contribution of positive and negative associations to the overall measure was investigated.

Design and Methods:

The implicit weight attitude was assessed using the Implicit Association Test. A Rasch model was used to identify which stimuli most affected the implicit measure. Explicit attitudes were assessed with a thin‐fat preference scale, and thermometer scales for both thin and fat people. The sample consists of 510 individuals categorized according to their BMI.

Results and Conclusion:

A significant implicit preference for thin relative to fat people was observed in all weight groups. In normal weight and obese individuals, the preference was mostly affected by positive stimuli (more easily associated with thin than with fat people). In underweight individuals, the preference was mostly guided by positive (more easily associated with thin than with fat people) and negative (more easily associated with fat than with thin people) stimuli. In overweight individuals, all stimuli contributed to the preference in a similar way. In all weight groups, the implicit weight attitude correlated with the explicit preference and/or the thin thermometer, whereas it never correlated with the fat thermometer. A pro‐thin bias was observed in normal weight and obese individuals, whereas both a pro‐thin and an anti‐fat bias were observed in underweight individuals. A clear preference for thin people relative to fat people was observed in overweight individuals. Therefore, uncritically interpreting the implicit preference for thin people as a sign of derogating fat people might be misleading.     

A novel type of highly negatively charged lipooligosaccharide from Pseudomonas stutzeri OX1 possessing two 4,6-O-(1-carboxy)-ethylidene residues in the outer core region.

Pseudomonas stutzeri OXI is a Gram-negative microorganism able to grow in media containing aromatic hydrocarbons. A novel lipo-oligosaccharide from P. stutzeri OX1 was isolated and characterized. For the first time, the presence of two moieties of 4,6-O-(1-carboxy)-ethylidene residues (pyruvic acid) was identified in a core region; these two residues were found to possess different absolute configuration. The structure of the oligosaccharide backbone was determined using either alkaline or acid hydrolysis. Alkaline treatment, aimed at recovering the complete carbohydrate backbone, was carried out by mild hydrazinolysis (de-O-acylation) followed by de-N-acylation using hot KOH. The lipo-oligosaccharide was also analyzed after acid treatment, attained by mild hydrolysis with acetic acid, to obtain information on the nature of the phosphate and acyl groups. The two resulting oligosaccharides were isolated by gel permeation chromatography, and investigated by compositional and methylation analyses, by MALDI mass spectrometry, and by 1H-, 31P- and 13C-NMR spectroscopy. These experiments led to the identification of the major oligosaccharide structure representative of core region-lipid A. All sugars are D-pyranoses and alpha-linked, if not stated otherwise. Based on the structure found, the hypothesis can be advanced that pyruvate residues are used to block elongation of the oligosaccharide chain. This would lead to a less hydrophilic cellular surface, indicating an adaptive response of P. sutzeri OX1 to a hydrocarbon-containing environment.     

Structural determination of the O-chain polysaccharide from Agrobacterium tumefaciens, strain DSM 30205.

Agrobacterium tumefaciens is a Gram-negative, phytopathogenic bacterium and is characterized by an unique mode of action on dicotyledonous plants: it is able to genetically modify the host, and because of this feature, it is used as a tool for transgenic plants. Many experiments have demonstrated that lipopolysaccharides (LPSs) play an important role for the disease development, as they are involved in the adhesion process of the bacterium on the plant cell wall. Despite the wealth of information on the role of LPS on phytopathogenesis, the present paper appears as the first report on the molecular primary structure of the O-chain produced from Agrobacterium. Its repeating unit was determined by means of chemical and spectroscopical analysis, and has the following structure: (3)-alpha-D-Araf-(1-->3)-alpha-l-Fucp-(1-->.     

Application of the nearest-neighbor Ising model to the helix-coil transition of polypeptides in mixed organic solvents.

Using the formalism of nearest-neighbor Ising model and assuming that the allowed states for a monomeric unity of a polypeptide chain in solutions containing strong acids are E (helix), C (coil), and CS (solvent-bonded coil), the partition function of the system was deduced analytically. Equations were obtained which permitted the prediction of the characteristic thermodynamic behavior of the helix–coil transition under these conditions. These equations were used to examine critically the possible correlations between experimental data obtained using different techniques. Particular attention was devoted to quantities called “transition enthalpies,” obtained from the slope of the transition curves at the point where the helix fraction is one-half (ΔH), or for measurements of the heat of solution of the polymer over the total range of solvent composition (ΔH), or from heat capacity measurements taken at various temperatures (ΔH). Literature data of ΔH(j = opt, sol, cal) for the system poly-γ-benzyl-L -glutamate in mixtures of dichloroacetic acid and 1,2-dichloroethane were carefully analyzed.     

The linkage between O-specific caryan and core region in the lipopolysaccharide of Burkholderia caryophylli is furnished by a primer monosaccharide

From the lipopolysaccharide (LPS) fraction of the plant-pathogenic bacterium Burkholderia caryophylli, the linkage between O-specific caryan and core region was characterised. The LPS fraction was first treated with 48% aqueous HF at 4 degrees C and successively with 1% acetic acid at 100 degrees C. A main oligosaccharide representing the carbohydrate backbone of the core region and a portion of the caryan (three unit of caryose) was isolated by high-performance anion-exchange chromatography. Compositional and methylation analyses, matrix-assisted laser desorption/ionisation mass spectrometry and 2D NMR spectroscopy identified the structure: [carbohydrate structure: see text]. The above residues are alpha-linked pyranose rings, if not stated otherwise. Hep is L-glycero-D-manno-heptose, Car is 4,8-cyclo-3,9-dideoxy-L-erythro-D-ido-nonose and Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid. This finding indicates that QuiNAc residue is the primer monosaccharide, which connects the core oligosaccharide to caryan O-chain.     

The structure of the phosphorylated carbohydrate backbone of the lipopolysaccharide of the phytopathogen bacterium Pseudomonas tolaasii

A novel core-lipid A backbone oligosaccharide was isolated and identified from the lipopolysaccharide fraction of the mushrooms pathogen bacterium Pseudomonas tolaasii. The oligosaccharide was obtained by alkaline treatment of the lipopolysaccharide fraction. Since the repeating unit of the O-antigen contained one residue of -->4)-alpha-l-GulpNAcAN, the hydrolysis was accompanied by beta-elimination on this residue and following depolymerization, producing a mixture of oligosaccharides. The complete structural elucidation showed the presence of a single core glycoform and was achieved by chemical analysis and by (1)H, (31)P, and (13)C NMR spectroscopy applying various 1D and 2D experiments. [structure: see text]. All sugars are alpha-d-pyranoses, if not stated otherwise. Hep is l-glycero-d-manno-heptose, Kdo is 3-deoxy-d-manno-oct-2-ulosonic acid, P is phosphate. QuiN and DeltaGulNA are present in nonstoichiometric amount.