Small-angle X-ray scattering studies of enzymes

Enzyme function requires conformational changes to achieve substrate binding, domain rearrangements, and interactions with partner proteins, but these movements are difficult to observe. Small-angle X-ray scattering (SAXS) is a versatile structural technique that can probe such conformational changes under solution conditions that are physiologically relevant. Although it is generally considered a low-resolution structural technique, when used to study conformational changes as a function of time, ligand binding, or protein interactions, SAXS can provide rich insight into enzyme behavior, including subtle domain movements. In this perspective, we highlight recent uses of SAXS to probe structural enzyme changes upon ligand and partner-protein binding and discuss tools for signal deconvolution of complex protein solutions.     

Non-destructive methods for demonstrating chemical changes in the rhizosphere II. Application of methods

Chemical changes in the rhizosphere of soil-grown plants are demonstrated by non-destructive techniques based on colour reactions. The following examples are given: FeIII reduction in the rhizosphere of a Hakea species, MnIV reduction in the rhizosphere of chikpea, complexation of Al in the rhizosphere of Norway spruce, and the activity of acid phosphatase in the rhizosphere of maize.     

Stoichiometric complexation of streptomyces subtilisin inhibitor and subtilisin

Subtilisin (Sbt) andStreptomyces subtilisin inhibitor (SSI) were analyzed either alone or together using sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). With all ratios of Sbt to SSI tested, the proteins formed a stoichiometric complex, and migrated abnormally at the top of the gel. Electroblotting and amino acid sequence analysis of the complex band showed both Sbt and SSI present at approximately equal molar ratios. When excess Sbt was present, it migrated as a free but still folded form slightly above the band corresponding to the complex. When excess SSI was present, it migrated as several species with molecular weights smaller than the intact form; in fact, the sequences of some of these species indicated that they lacked different amounts of N-terminal and possibly C-terminal residues.     

Preparation of a chiral ferrocenyl derivative containing a biphenyl unit via nickel-mediated homocoupling

Ferrocenyl derivative 2, which possesses a biphenyl unit, was prepared by nickel(0)-mediated homocoupling of a chiral ferrocenyloxazoline. Compound 2 exists as an equilibrium mixture of two diastereomers, in a ratio dependent on temperature and solvent, due to the partially hindered rotation around biaryl bond. Upon complexation with Cu(I) or Zn(II), complete fixation of the stereogenic axis was obtained and a single stable atropoisomer was formed, whose absolute configuration was assigned on the basis of its NOESY spectrum. The 2-Cu(I) complex was used as catalyst in the cyclopropanation of styrene.     

Microscale heterogeneity of acidity related stress-parameters in the soil solution of a forested cambic podzol

Acid related stress in soils might be caused by high concentrations of H⁺ and Al³⁺ in soil solution. Sampling of soil solution so far integrates over a relatively large soil volume, in the range of dm³. In order to study the microscale heterogeneity of acidity related stress-parameters the soil profile of a podzolic cambisol was covered by a 10×6 matrix of micro suction cups with a grid distance of 2 cm. The soil solution collected at 10 sampling events was analyzed for free cations and anions by capillary electrophoresis and for total metal content by a micro injection technique on ICP-OES. pH and UV absorption were also measured.There was a general trend of increasing pH and decreasing UV absorption with increasing soil depth, however without a clear correlation of concentration isolines to soil horizon borders. The latter was also true for total Al (Al_tot) and Al³⁺, with the exception of the soil horizon border A_he/B_h,which was very well reflected by Al³⁺ and also by the fraction of bound Al. In the A_he horizon less than 30%, in deeper mineral soil less than 50% of Al_tot were present as free Al³⁺. This fact is critical when calculating Ca/Al ratios as a stress parameter, because total metal content measured by ICP clearly overestimates the risk of root damage, even in deeper horizons of acid forest soils, where organic complexation of Al is of minor importance. The heterogeneity of soil solution chemistry and toxicity parameters on the cm-scale was found to be significant, for example with gradients of more than 0,5 pH-units within 2 cm. Because plant roots also experience soil on a microscale, high resolution investigations of soil solution chemistry offer a new approach for looking at the chemical environment relevant for root growth and plant nutrition.     

Modeling of Selforganizing Systems

The concept of self-assembly developed for the construction of topologically complex molecules such as [2]-catenanes and rotaxanes is based on non-covalent interactions between constituent parts. High product yields are explained by favourable orientations of intermediates. These intermediates are found to be 2 - 9 kcal/mol lower in energy than the reactand ground states and the recognition process could be reproduced by semiempirical calculations using the PM3 Hamiltonian. Cyclobis(paraquat-p-phenylene) (1 ⁴⁺), which is known for its extraordinary capability to form charge-transfer complexes, plays an important role. The conformational analysis of the hypersurface of (1 ⁴⁺) and other compounds was performed using the MM2 force field. The concept of self-organization uses electron accepting hosts like 1 ⁴⁺ as well as donating hosts like crown ethers. Therefore the study was extended on donating hosts and on their capability to form catenanes. These very interesting compounds show stabilization energies of about 36 kcal/mol. Electronic Supplementary Material available.     

Synthesis and Enantiomeric Recognition Studies of Optically Active Pyridino‐Crown Ethers Containing an Anthracene Fluorophore Unit

Novel enantiopure pyridino‐18‐crown‐6 ether‐based sensor molecules containing an anthracene fluorophore unit were synthesized. Their enantiomeric recognition abilities toward the enantiomers of 1‐phenylethylamine hydrogen perchlorate (PhEt), 1‐(1‐naphthyl)ethylamine hydrogen perchlorate (NapEt), phenylglycine methyl ester hydrogen perchlorate (PhgOMe), and phenylalanine methyl ester hydrogen perchlorate (PheOMe) were examined in acetonitrile using fluorescence spectroscopy. The sensor molecules showed appreciable enantiomeric recognition toward the enantiomers of NapEt, PhEt, and PhgOMe. The highest enantioselectivity was found in the case of crown ether containing isobutyl groups in the macroring and the enantiomers of NapEt. Chirality 28:562–568, 2016. © 2016 Wiley Periodicals, Inc.     

The Effects of Bacterial Surface Adsorption and Exudates on HgO Precipitation

The effects of nonmetabolic bacterial cell wall adsorption and the presence of bacterial exudates on the precipitation of mineral phases from solution is not well constrained experimentally. In this study, we measured the extent of Hg(II) removal from solution, in the presence and absence of nonmetabolizing cells of Bacillus subtilis in both Cl-free and Cl-bearing systems with Hg concentrations ranging from undersaturation to supersaturation with respect to montroydite [HgO_(s)]. Total Hg molalities ranged from 10^?5.00 to 10^?2.00 M at pH 4.50 and 7.00; the ionic strength of the experiments was kept constant using 0.01 M NaClO₄, and the wet mass of bacteria was held constant at 5 g/L for each biotic experiment.

The biotic systems exhibited enhanced Hg(II) removal from solution relative to the abiotic controls in undersaturated conditions. However, thermodynamic modeling of the experimental systems strongly suggests that all of this Hg removal can be ascribed to Hg adsorption onto cell envelope functional groups. There was no evidence for enhanced Hg removal due to precipitation in bulk solutions that were undersaturated with respect to the solid phase. Under the highest total Hg concentrations studied in both the Cl-free and Cl-bearing systems, bacteria inhibit precipitation, maintaining high concentrations of Hg in solution. Cell-free, exudate-bearing control experiments suggest that aqueous complexation between Hg and the bacterially-produced exudates accounts for at least some of the precipitation inhibition. However, a comparison of total available binding sites on the exudates with the concentration of Hg in solution suggests that aqueous complexation alone can not account for the observed elevated final aqueous Hg concentrations in solution, and that the exudates likely exert a kinetic inhibition on the precipitation reaction as well.     

Distribution of polyphosphates in cell-compartments of Chlorella fusca as measured by 31P-NMR-spectroscopy

In suspensions of the green alga Chlorella fusca the influence of high pH and high ethylene-diamine-tetraacetic acid concentrations in the external medium, of French-press and perchloric acid extraction of the cells and of alkalization of the intracellular pH on the polyphosphate signal in ³¹P-nuclear magnetic resonance (³¹P NMR) spectra was investigated.The results show that part of the polyphosphates of asynchronous Chlorella cells are located outside the cytoplasmic membrane and complexed with divalent metal-ions. These polyphosphates are tightly bound to the cell wall and/or the cytoplasmic membrane and are not susceptible to hydrolyzation by strong acid at room temperature, in contrast to the intracytoplasmic polyphosphates.Upon alkalization of the internal pH of Chlorella cells, polyphosphates, previously not visible in the spectra become detectable by ³¹P-NMR-spectroscopy. ³¹P-NMR spectroscopic monitoring of polyphosphates during gradual alkalization of the extra-and intracellular space is proposed as a quick method for the estimation of the cellular polyphosphate content and distribution.Abbreviations CCCP Carbonylcyanide-m-chlorophenyl-hydrazone - NTP/NDP Nucleotide triphosphate/-diphosphate - PCA Perchloric acid - ³¹P-NMR ³¹P-nuclear magnetic resonance - PolyP polyphosphates - PP₁, PP₂, PP₃ terminal, second and third phosphate residue of polyphosphates, respectively - PP₄ core phosphate residues of polyphosphates     

The toxicity of cadmium to barley plants as affected by complex formation with humic acid

An ‘alternating solution’ culture method was used to study the effects of chloride ions and humic acid (HA) on the uptake of cadmium by barley plants. The plants were transferred periodically between a nutrient solution and a test solution containing one of four levels of HA (0, 190, 569 or 1710 μg cm⁻³) and one of five levels of Cd (0, 0.5, 1.0, 2.5 or 5.0 μg cm⁻³) in either a 0.006M NaNO₃ or 0.006M NaCl medium. Harvest and analysis of shoots and roots was after nineteen days. The distribution of Cd in the test solutions between Cd²⁺, CdCl⁺ and HA-Cd was determined in a separate experiment by dialysis equilibrium. In the nitrate test solutions Cd uptake was clearly controlled by Cd²⁺ concentration and was therefore reduced by HA complex formation. In the absence of HA, chloride suppressed Cd uptake indicating that Cd²⁺ was the preferred species. However complex formation with Cl⁻ enhanced uptake when HA was present because of an increase in the concentration of inorganic Cd species relative to the nitrate system. The ratio root-Cd/shoot-Cd remained at about 10 across a wide range of shoot-Cd concentrations, from about 3 μg g⁻¹ (sub-toxic) up to 85 μg g⁻¹ (80% yield reduction). The ability of the barley plants to accumulate ‘non-toxic’ Cd in their roots was thus very limited. Humic acid also had no effect on Cd translocation within the plant and the root/shoot weight ratio did not vary with any treatment. At shoot-Cd concentrations in excess of 50 μg g⁻¹, K, Ca, Cu and Zn uptake was reduced, probably the result of root damage rather than a specific ion antagonism. The highest concentration of HA also lowered Fe and Zn uptake and there was a toxic effect with increasing HA concentration at Cd=0. However the lowest HA level, comparable with concentrations found in mineral soil solutions, only reduced yield (in the absence of Cd) by <5% while lowering Cd uptake across the range of Cd concentrations by 66%–25%.