A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral Ln III tag, referred to as C12, is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues.Loaded with Tb III or Tm III ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron-electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the Tb III complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.
The design of molecular receptors that bind and sense anions in biologically relevant aqueous solutions is a key challenge in supramolecular chemistry. The recognition of inorganic phosphate is particularly challenging...
Peroxynitrite (ONOO À ) is a powerful and short-lived oxidant formed in vivo, which can react with most biomolecules directly. To fully understand the roles of ONOO À in cell biology, improved methods for the selective detection and real-time analysis of ONOO À are needed. We present a water-soluble, luminescent europium(III) probe for the rapid and sensitive detection of peroxynitrite in human serum, living cells and biological matrices. We have utilised the long luminescence lifetime of the probe to measure ONOO À in a time-resolved manner, effectively avoiding the influence of autofluorescence in biological samples. To demonstrate the utility of the Eu(III) probe, we monitored the production of ONOO À in different cell lines, following treatment with a cold atmospheric plasma device commonly used in the clinic for skin wound treatment.
Members of the SLC26 family constitute a conserved family of anion transport proteins, which encompasses uncoupled transporters with channel-like properties, coupled exchangers and motor proteins. Among the eleven paralogs in humans, several participate in the secretion of bicarbonate in exchange with chloride and thus play an important role in pH homeostasis. Previously, we have elucidated the structure of murine SLC26A9 and defined its function as an uncoupled chloride transporter (Walter, Sawicka, & Dutzler, 2019). Here we have determined the structure of the closely related human transporter SLC26A6 and characterized it as a strictly coupled exchanger of chloride with either bicarbonate or oxalate. The structure defines an inward-facing conformation of the protein that generally resembles known structures of SLC26A9. The altered anion selectivity between both paralogs is a consequence of a remodeled ion binding site located in the center of a mobile unit of the membrane-inserted domain, which also accounts for differences in the coupling mechanism.
Non-thermal plasma (NTP), an ionized gas generated at ambient pressure and temperature, has been an emerging technology for medical applications. Through controlled delivery of reactive oxygen and nitrogen species (ROS/RNS), NTP can elicit hormetic cellular responses, thus stimulating broad therapeutic effects. To enable clinical translation of the promising preclinical research into NTP therapy, a deeper understanding of NTP interactions with clinical substrates is profoundly needed. Since NTP-generated ROS/RNS will inevitably interact with blood in several clinical contexts, understanding their stability in this system is crucial. In this study, two medically relevant NTP delivery modalities were used to assess the stability of NTP-generated ROS/RNS in three aqueous solutions with increasing organic complexities: phosphate-buffered saline (PBS), blood plasma (BP), and processed whole blood. NTP-generated RNS collectively (NO2−, ONOO−), H2O2, and ONOO− exclusively were analyzed over time. We demonstrated that NTP-generated RNS and H2O2 were stable in PBS but scavenged by different components of the blood. While RNS remained stable in BP after initial scavenging effects, it was completely reduced in processed whole blood. On the other hand, H2O2 was completely scavenged in both liquids over time. Our previously developed luminescent probe europium(III) was used for precision measurement of ONOO− concentration. NTP-generated ONOO− was detected in all three liquids for up to at least 30 seconds, thus highlighting its therapeutic potential. Based on our results, we discussed the necessary considerations to choose the most optimal NTP modality for delivery of ROS/RNS to and via blood in the clinical context.
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