Kinetics of rapid covalent bond formation of aniline with humic acid: ESR investigations with nitroxide spin labels

ABSTRACT The bioavailability of many soil contaminants depends on their interaction with the soil organic matter. The paper presents a new approach of using stable paramagnetic spin labels for investigating the kinetics of covalent binding of specific xenobiotic functional groups with humic acids, a major organic matter fraction. Leonardite humic acid (LHA) was incubated with the nitroxide spin labels amino-TEMPO (4-amino-2,2,6,6-Tetramethylpiperidin-1-oxyl) and anilino-NO (2,5,5-Trimethyl-2-(3-aminophenyl)pyrrolidin-1-oxyl), respectively, which contain an aliphatic or aromatic functionality susceptible to interaction with LHA. Electron spin resonance (ESR) spectra of LHA samples without and with the enzyme laccase were recorded at X-band frequency (9.43 GHz) at room temperature and neutral pH. Binding was detected by a pronounced broadening of the spectral lines after incubation of LHA for both spin labels. The development of a broad signal component in the spectrum of anilino-NO indicated the immobilization due to strong binding of the aniline group. The reorientational correlation time of bound anilino-NO is more than two orders of magnitude greater than that of the free label. The ratio of the amount of bound to the unbound species was used to determine the kinetics of the covalent bond formation. Reaction rate constants of 0.16 min-1 and 0.01 min-1 were determined corresponding to half-times of 4.3 min and 69.3 min, respectively. Treatment of LHA with laccase enhanced the amount of the reacting anilino-NO species by a factor of 7.6, but left the reaction rate unaltered. Oxidative radical coupling was excluded by using the spin trap agent n-tert-butyl-alpha-phenylnitrone

Addressing the optimal silver content in bioactive glass systems in terms of BSA adsorption

Bioactive glasses doped with silver are aimed to minimize the risk of microbial contamination, and therefore, the influence of silver on the bioactive properties is an intense investigated task. However, the information related to the role played by silver, when added to the bioactive glass composition, on the biocompatibility properties is scarce. This aspect is essential as long as the silver content can influence the blood protein adsorption onto glass surface, affecting thus the material biocompatibility. Therefore, from the perspective of the biocompatibility standpoint, the finding of an optimal silver content in a bioactive glass is an extremely important issue. In this study, silver doped bioactive glasses were prepared by melt-derived technique, which eliminates the pores influence in the protein adsorption process. The obtained glasses were characterized by X-ray diffraction, UV-vis, X-ray Photoelectron (XPS) and Fourier Transform Infrared (FT-IR) spectroscopy, and afterwards they were investigated in terms of protein adsorption. Both UV-vis and XPS spectroscopy revealed the presence of Ag+ ions in all silver containing samples. By increasing the silver content, metallic Ag0 appears, the highest amount being observed for the sample with 1 mol% AgO2. Electron Paramagnetic Resonance measurements evidenced that the amount of spin labeled serum albumin attached on the surface increases with the silver content. The results obtained by analyzing the information derived from Atomic Force Microscopy and FT-IR measurements indicate that the occurrence of metallic Ag0 in the samples structure influences the secondary structure of the adsorbed protein. Based on the results derived from the protein response upon interaction with the investigated glass calcium-phosphate based system it was determined the optimal silver oxide concentration for which the secondary structure of the adsorbed protein is similar with that of the free one. This concentration was found to be 0.5 mol%

Characterization of multifunctional β-NaEuF4/NaGdF4 core–shell nanoparticles with narrow size distribution

We characterized NaEuF4/NaGdF4 core–shell nanoparticles

Interconversion between bound and free conformations of LexA orchestrates the bacterial SOS response

The bacterial SOS response is essential for the maintenance of genomes, and also modulates antibiotic resistance and controls multidrug tolerance in subpopulations of cells known as persisters. In Escherichia coli, the SOS system is controlled by the interplay of the dimeric LexA transcriptional repressor with an inducer, the active RecA filament, which forms at sites of DNA damage and activates LexA for self-cleavage. Our aim was to understand how RecA filament formation at any chromosomal location can induce the SOS system, which could explain the mechanism for precise timing of induction of SOS genes. Here, we show that stimulated self-cleavage of the LexA repressor is prevented by binding to specific DNA operator targets. Distance measurements using pulse electron paramagnetic resonance spectroscopy reveal that in unbound LexA, the DNA-binding domains sample different conformations. One of these conformations is captured when LexA is bound to operator targets and this precludes interaction by RecA. Hence, the conformational flexibility of unbound LexA is the key element in establishing a co-ordinated SOS response. We show that, while LexA exhibits diverse dissociation rates from operators, it interacts extremely rapidly with DNA target sites. Modulation of LexA activity changes the occurrence of persister cells in bacterial populations

Phosphorylation of a membrane curvature–sensing motif switches function of the HOPS subunit Vps41 in membrane tethering

An AP-3–binding site required for vesicle–vacuole fusion is masked when Vps41 is associated with highly curved membranes, such as endosomes, but is exposed at membranes with lower curvature, such as vacuoles, because of phosphorylation of the membrane-binding motif

Simulation vs. Reality: A Comparison of In Silico Distance Predictions with DEER and FRET Measurements

Site specific incorporation of molecular probes such as fluorescent- and nitroxide spin-labels into biomolecules, and subsequent analysis by Förster resonance energy transfer (FRET) and double electron-electron resonance (DEER) can elucidate the distance and distance-changes between the probes. However, the probes have an intrinsic conformational flexibility due to the linker by which they are conjugated to the biomolecule. This property minimizes the influence of the label side chain on the structure of the target molecule, but complicates the direct correlation of the experimental inter-label distances with the macromolecular structure or changes thereof. Simulation methods that account for the conformational flexibility and orientation of the probe(s) can be helpful in overcoming this problem. We performed distance measurements using FRET and DEER and explored different simulation techniques to predict inter-label distances using the Rpo4/7 stalk module of the M. jannaschii RNA polymerase. This is a suitable model system because it is rigid and a high-resolution X-ray structure is available. The conformations of the fluorescent labels and nitroxide spin labels on Rpo4/7 were modeled using in vacuo molecular dynamics simulations (MD) and a stochastic Monte Carlo sampling approach. For the nitroxide probes we also performed MD simulations with explicit water and carried out a rotamer library analysis. Our results show that the Monte Carlo simulations are in better agreement with experiments than the MD simulations and the rotamer library approach results in plausible distance predictions. Because the latter is the least computationally demanding of the methods we have explored, and is readily available to many researchers, it prevails as the method of choice for the interpretation of DEER distance distributions

Kissing G Domains of MnmE Monitored by X-Ray Crystallography and Pulse Electron Paramagnetic Resonance Spectroscopy

The authors of this research article demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle, and show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein. These movements are of crucial importance for understanding the mechanistic principles of this GAD

Molecular mechanisms of gene regulation studied by site-directed spin labeling.

The technique of site-directed spin labeling using cysteine substitution mutagenesis followed by modification of the sulfhydryl group with a nitroxide reagent is emerging as a valuable alternative for the determination of protein folds and conformational changes in a variety of systems. The incorporation of pairs of nitroxides allows determination of intramolecular distances and distance changes with a spatial resolution at the level of the backbone fold under conditions relevant to function. The methodology of electron paramagnetic resonance spectral data acquisition and interpretation is reviewed with studies on conformational changes of Tet repressor (TetR) and the human immunodeficiency virus type 1 reverse transcriptase (RT) on interaction with nucleic acid substrates or inhibitors in solution. A twisting motion of the DNA reading heads of TetR on induction by tetracycline (tc) is observed in solution by changes of the interspin distances between interacting nitroxides at positions 22/22(') or 47/47('). Spin-label side chains located near the tc-binding pocket or at position 202 indicate different conformations for the tc- and DNA-complexed repressor also in the core of the protein. Interspin distances between spin-labeled residue positions 24 and 287 in the fingers and the thumb domains of RT complexed with dsDNA or a pseudoknot RNA in solution were found to agree with the respective crystal data of the so-called open and closed conformations. For the unliganded RT a temperature-dependent equilibrium between these two states is observed