Stabilizing Nitroxide Spin Labels for Structural and Conformational Studies of Biomolecules by Maleimide Treatment.

Nitroxide (NO) spin radicals are effective in characterizing structures, interactions and dynamics of biomolecules. The EPR applications in cell lysates or intracellular milieu require stable spin labels, but NO radicals are unstable in such conditions. We showed that the destabilization of NO radicals in cell lysates or even in cells is caused by NADPH/NADH related enzymes, but not by the commonly believed reducing reagents such as GSH. Maleimide stabilizes the NO radicals in the cell lysates by consumption of the NADPH/NADH that are essential for the enzymes involved in destabilizing NO radicals, instead of serving as the solo thiol scavenger. The maleimide treatment retains the crowding properties of the intracellular components and allows to perform long-time EPR measurements of NO labeled biomolecules close to the intracellular conditions. The strategy of maleimide treatment on cell lysates for the EPR applications has been demonstrated on double electron-electron resonance (DEER) measurements on a number of NO labeled protein samples. The method opens a broad application range for the NO labeled biomolecules by EPR in conditions that resemble the intracellular milieu.

Simultaneous Quantification of Biothiols and Deciphering Diverse GSH Stability in Different Live Cells by 19F-Tag.

GSH, Cys, Hcy, and H2S are important biothiols and play important roles in the living systems. Quantitative and simultaneous determination of these biothiols under physiological conditions is still a challenge. Herein, we developed an effective 19F-reactive tag that readily interacts with these four biothiols for the generation of stable thioether products that have distinguishable 19F-chemical shifts. These thioester compounds encode the characteristic fingerprint profiles of each biothiols, allowing one to simultaneously quantify and determine these biothiols by 1D 19F NMR spectroscopy. The intra-/extracellular GSH in live cells was assessed by the established strategy, and remarkable variations in the GSH stability were determined between the normal mammalian cells and cancer cells. It is notable that GSH hydrolyzes efficiently in the out-membrane of the cancer cells and the lysates. In contrast, GSH remains stable in the tested normal cells.

Rigid, Highly Reactive and Stable DOTA-like Tags Containing a Thiol-Specific Phenylsulfonyl Pyridine Moiety for Protein Modification and NMR Analysis*.

Site specific installation of a paramagnetic ion with magnetic anisotropy in a biomolecule generates valuable structural restraints, such as pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs). These paramagnetic effects can be used to characterize the structures, interactions and dynamics of biological macromolecules and their complexes. Two single-armed DOTA-like tags, BrPSPy-DO3M(S)A-Ln and BrPSPy-6M-DO3M(S)A-Ln, each containing a thiol-specific reacting group, that is, a phenylsulfonyl pyridine moiety, are demonstrated as rigid, reactive and stable paramagnetic tags for protein modification by formation of a reducing resistant thioether bond between the protein and the tag. The two tags present high reactivity with the solvent exposed thiol group in aqueous solution at room temperature. The introduction of Br at the meta-position in pyridine enhances the reactivity of 4-phenylsulfonyl pyridine towards the solvent exposed thiol group in a protein, whereas the ortho-methyl group in pyridine increases the rigidity of the tag in the protein conjugates. The high performance of these two tags has been demonstrated in different cysteine mutants of ubiquitin and GB1. The high reactivity and rigidity of these two tags can be added in the toolbox of paramagnetic tags suitable for the high-resolution NMR measurements of biological macromolecules and their complexes.

Four novel species of Pleurotheciaceae collected from freshwater habitats in Jiangxi Province, China.

During an investigation of fungal diversity from freshwater environments in different regions in Jiangxi Province, China, four interesting species were collected. Morphology coupled with combined gene analysis of an ITS, LSU, SSU, and rpb2 DNA sequence data showed that they belong to the family Pleurotheciaceae. Four new species, Pleurotheciella ganzhouensis, Pla. irregularis, Pla. verrucosa, and Pleurothecium jiangxiense are herein described. Pleurotheciella ganzhouensis is characterized by its capsule-shaped conidia and short conidiophores, while Pla. irregularis has amorphous conidiophores and 3-septate conidia. Pleurotheciella verrucosa has cylindrical or verrucolose conidiogenous cells, 1-septate, narrowly fusiform, meniscus or subclavate conidia. Pleurothecium jiangxiense characterized in having conidiogenous cells with dense cylindrical denticles and short conidiophores. Pleurothecium obovoideum was transferred to Neomonodictys based on phylogenetic evidence. All species are compared with other similar species and comprehensive descriptions, micrographs, and phylogenetic data are provided.

Real-Time Monitoring of the Level and Activity of Intracellular Glutathione in Live Cells at Atomic Resolution by 19F-NMR.

Visualization and quantification of important biomolecules like glutathione (GSH) in live cells are highly important. The existing methods are mostly from optical detection and lack of atomic resolution on the activity of GSH. Here, we present a sensitive 19F-NMR method to quantify real-time variations of GSH in live cells in a reversible manner. This NMR method prevents extracellular leakage and irreversible consumption of intracellular GSH during the detection. The high performance of the reactive 19F-probe enables accurate determination of intracellular GSH content at atomic resolution, from which information on GSH variations with respect to the extracellular and intracellular conditions can be inferred. In addition, we demonstrate the applicability of this NMR method to quantify the GSH levels between different live cell lines and to disclose the distinct differences between the intracellular environment and cell lysates. We foresee the application of 19F-NMR to monitor real-time variations of intracellular GSH levels in relation to GSH-involved central cellular processes.

Simultaneous identification and quantification of amino acids in biofluids by reactive 19F-tags.

A robust method to identify and quantify amino acids close to physiological conditions by 1D 19F NMR was established. Each 19F-derivatized amino acid has its characteristic chemical-shift profile that is readily identified in the mixture of amino acids or in biofluids including fetal bovine serum and cell lysates. The method shows great potential in metabolomics and biochemical analysis.