No abstract

In this study, a series of magnetic ionic liquids (MILs) were investigated for the extraction and preconcentration of bacteria from aqueous samples. By dispersing small volumes (e.g., 15 μL) of MIL within an aqueous cell suspension, bacteria were rapidly extracted and isolated using a magnetic field. Of the seven hydrophobic MILs examined, the trihexyl(tetradecyl)phosphonium Ni(II) hexafluoroacetylacetonate ([P][Ni(hfacac)]) MIL exhibited the greatest enrichment of viable Escherichia coli K12 when coupled with microbiological culture as the detection method. The MIL-based strategy was applied for the preconcentration of E. coli from aqueous samples to obtain enrichment factors (E ) as high as 44.6 in less than 10 min. The MIL extraction approach was also interfaced with polymerase chain reaction (PCR) amplification where the positive detection of E. coli was achieved with the [P][Co(hfacac)], [P][Ni(hfacac)], [P][Dy(hfacac)], and [P][Nd(hfacac)] MILs. While direct sampling of an aqueous cell suspension at a concentration of 1.68 × 10 colony-forming units (CFUs) mL yielded no amplicon when subjected to PCR, extraction of the sample with the [P][Ni(hfacac)] MIL under optimized conditions provided sufficient enrichment of E. coli for amplicon detection. Importantly, the enrichment of bacteria using the Ni(II)-, Co(II)-, and Dy(III)-based MILs was compatible with real-time quantitative PCR amplification to dramatically improve sample throughput and lower detection limits to 1.0 × 10 CFUs mL. The MIL-based method is much faster than existing enrichment approaches that typically require 24-h cultivation times prior to detection and could potentially be applied for the preconcentration of a variety of Gram-negative bacteria from aqueous samples. Graphical abstract Magnetic ionic liquid solvents rapidly preconcentrate viable E. coli cells for unambiguous pathogen detection using microbiological culture and qPCR.

Protein-protein interactions and the complexes thus formed are critical elements in a wide variety of cellular events that require an atomic-level description to understand them in detail. Such complexes typically constitute challenging systems to characterize and drive the development of innovative biophysical methods. NMR spectroscopy techniques can be applied to extract atomic resolution information on the binding interfaces, intermolecular affinity, and binding-induced conformational changes in protein-protein complexes formed in solution, in the cell membrane, and in large macromolecular assemblies. Here we discuss experimental techniques for the characterization of protein-protein complexes in both solution NMR and solid-state NMR spectroscopy. The approaches include solvent paramagnetic relaxation enhancement and chemical shift perturbations (CSPs) for the identification of binding interfaces, and the application of intermolecular nuclear Overhauser effect spectroscopy and residual dipolar couplings to obtain structural constraints of protein-protein complexes in solution. Complementary methods in solid-state NMR are described, with emphasis on the versatility provided by heteronuclear dipolar recoupling to extract intermolecular constraints in differentially labeled protein complexes. The methods described are of particular relevance to the analysis of membrane proteins, such as those involved in signal transduction pathways, since they can potentially be characterized by both solution and solid-state NMR techniques, and thus outline key developments in this frontier of structural biology.

AlkB homolog 5 (Alkbh5) is one of nine members of the AlkB family, which are nonheme Fe 2þ /a-ketoglutaratedependent dioxygenases that catalyze the oxidative demethylation of modified nucleotides and amino acids. Alkbh5 is highly selective for the N 6 -methyladenosine modification, an epigenetic mark that has spawned significant biological and pharmacological interest because of its involvement in important physiological processes, such as carcinogenesis and stem cell differentiation. Herein, we investigate the structure and dynamics of human Alkbh5 in solution. By using 15 N and 13 C methyl relaxation dispersion and 15 N-R 1 and R 1r NMR experiments, we show that the active site of apo Alkbh5 experiences conformational dynamics on multiple timescales. Consistent with this observation, backbone amide residual dipolar couplings measured for Alkbh5 in phage pf1 are inconsistent with the static crystal structure of the enzyme. We developed a simple approach that combines residual dipolar coupling data and accelerated molecular dynamics simulations to calculate a conformational ensemble of Alkbh5 that is fully consistent with the experimental NMR data. Our structural model reveals that Alkbh5 is more disordered in solution than what is observed in the crystal state and undergoes breathing motions that expand the active site and allow access to a-ketoglutarate. Disordered-to-ordered conformational changes induced by sequential substrate/cofactor binding events have been often invoked to interpret biochemical data on the activity and specificity of AlkB proteins. The structural ensemble reported in this work provides the first atomic-resolution model of an AlkB protein in its disordered conformational state to our knowledge.

N-terminal fusion of the N-terminal domain of bacterial enzyme I facilitates N-terminal fusion of the N-terminal domain of bacterial enzyme I facilitates recombinant expression and purification of the human RNA demethylases FTO recombinant expression and purification of the human RNA demethylases FTO and Alkbh5 and Alkbh5