Binding States of Protein–Metal Complexes in Cells

The identification of endogenous proteins as well as their binding to metal ions in living cells is determined by combining pulsed electrophoretic separations with nanoelectrospray ionization followed by mass spectrometric detection. This approach avoids problems resulting from the complicated cellular environment. In this manner, we demonstrate the rapid identification (300 ms or less) of intact proteins from living E. coli cells including the complexation of calmodulin with calcium ion. The latter showed different binding states from those observed in in vitro studies. These observations also reveal in vitro measurements do not necessarily represent the actual situation in living cells. We conclude that the attempted in situ measurement of intracellular proteins with minimal sampling processes should be preferred

The Reaction of Arsenite with Proteins Relies on Solution Conditions

Arsenic is a biologically interesting element with both antitumor and carcinogenic effects. Zinc finger proteins (ZFPs) have been confirmed to be the cellular targets of arsenite; however, arsenite inhibits ZFPs much less efficiently in vitro than in vivo. The molecular mechanism of this difference is unknown. In this work, we found that the reaction of arsenite with ZFPs relies on the presence of small biomolecules such as glutathione (GSH), histidine, and cysteine (Cys). The weak acidity also enhances the reaction. Further study shows that the coordination of zinc is much more susceptible than that of arsenic to these solution conditions, which enhance the competition of arsenic. Notably, different from C₃H-type ZFPs, the C₂H₂-type ZFPs are more significantly influenced by the presence of thiol-containing molecules in the reaction. GSH and Cys can facilitate the reaction by participation of the coordination to As­(III) together with C₂H₂-type ZFPs. Consequently, the reactions are promoted both thermodynamically and kinetically via the formation of ternary complexes GSH-As-ZFP or Cys-As-ZFP. These results indicate that the reactions between arsenite and proteins are considerably modulated by environments such as the small biomolecules and the acidity of the solution. This finding clarifies the discrepancy observed in the reactions of arsenite in vitro versus in cells, and provides an insight into the molecular mechanism of arsenite

Interaction between Platinum Complexes and the C‑Terminal Motif of Human Copper Transporter 1

Human copper transporter 1 (hCTR1) facilitates the cellular uptake of cisplatin, and the extracellular N-terminal domain has been proven to coordinate to platinum drugs. It has been reported that the intracellular C-terminal motif is crucial for the function of hCTR1 in cisplatin influx. In this work, we conduct reactions of the intracellular motif with platinum drugs. The octapeptide from the C-terminal domain of hCTR1 is used, and the reactions are investigated using ultraviolet, high-performance liquid chromatography, electrospray ionization mass spectrometry, and nuclear magnetic resonance spectroscopy. Results show that the C8 peptide is highly reactive to cisplatin and oxaliplatin, and the -HCH sequence is the most favorable binding site of platinum agents. Cisplatin first binds to the cysteine residues in the reaction with the C8 peptide. The ammine ligand, even trans to a thiol ligand, can remain coordinated in platination adducts for a >12 h reaction. Intramolecular platinum migration was observed in the C8 peptide, and the ammine ligands remain coordinated to platinum during this process. This result indicates that hCTR1 can transfer cisplatin in the active form through a <i>trans</i> chelation process. These findings provide insight into the mechanism of the C-terminus of hCTR1 in the transfer of platinum drugs from the trimeric pore of hCTR1 to the cytoplasm

A General Chemiluminescence Strategy for Measuring Aptamer–Target Binding and Target Concentration

Although much effort has been made for studies on aptamer–target interactions due to promising applications of aptamers in biomedical and analytical fields, measurement of the aptamer–target binding constant and binding site still remains challenging. Herein, we report a sensitive label-free chemiluminescence (CL) strategy to determine the target concentration and, more importantly, to measure the target–aptamer binding constant and binding site. This approach is suitable for multiple types of targets, including small molecules, peptides, and proteins that can enhance the CL initiated by <i>N</i>-(amino­butyl)-<i>N</i>-ethyliso­luminol functionalized gold colloids, making the present method a general platform to investigate aptamer–target interactions. This approach can achieve extremely high sensitivity with nanogram samples for measuring the target–aptamer binding constant. And the measurement could be rapidly performed using a simple and low-cost CL system. It provides an effective tool for studying the binding of biologically important molecules to nucleic acids and the selection of aptamers. Besides, we have also discovered that the 14-mer aptamer fragment itself split from the ATP-binding aptamer could selectively capture ATP. The binding constant, site, and conformation between ATP and the 14-mer aptamer fragment were obtained using such a novel CL strategy and molecular dynamic simulation