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One of the mechanisms by which toxic metal ions interfere with cellular functions is ionic mimicry, where they bind to protein sites in lieu of native metals Ca2+ and Zn2+. The influence of crowded intracellular environments on these interactions is not well understood. Here, we demonstrate the application of in-cell and lysate NMR spectroscopy to obtain atomic-level information on how a potent environmental toxin cadmium interacts with its protein targets. The experiments, conducted in intact E. coli cells and their lysates, revealed that Cd2+ can profoundly affect the quinary interactions of its protein partners, and can replace Zn2+ in both labile and non-labile protein structural sites without significant perturbation of the membrane binding function. Surprisingly, in crowded molecular environments Cd2+ can effectively target not only all-sulfur and mixed sulfur/nitrogen but also all-oxygen coordination sites. The sulfur-rich coordination environments show significant promise for bioremedial applications, as demonstrated by the ability of the designed protein scaffold α3DIV to sequester intracellular cadmium. Our data suggests that in-cell NMR spectroscopy is a powerful tool for probing interactions of toxic metal ions with their potential protein targets, and for the assessment of potency of sequestering agents.more » « lessFree, publicly-accessible full text available November 5, 2024
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Many biological macromolecules rely on metal ions to maintain structural integrity and control their regulatory function. In biological fluids, detection and identification of metal ions requires sensitive analytical tools with clear readouts. In this work, we sought to investigate the potential of solution Nuclear Magnetic Resonance (NMR) spectroscopy to analyze metal ion solutions and mixtures. To enable 1H NMR detection, we prepared the complexes of eight metal ions with the chelating agent, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). The 1H NMR spectra were collected for BAPTA samples as a function of metal ion concentrations. The analysis of NMR data revealed that all metal ions with a notable exception of Mg2+ bind BAPTA with high affinities and form complexes with 1:1 metal-to-chelator stoichiometry. Both methylene and aromatic regions of the BAPTA 1H NMR spectra experience significant changes upon the metal ion complex formation. We identified the spectroscopic signatures of trivalent and paramagnetic ions and demonstrated that the binary Zn2+/Pb2+ metal ion mixture can be successfully analyzed by NMR. We conclude that complexation with BAPTA followed by the 1H NMR analysis is a sensitive method to detect and identify both nutritive and xenobiotic metal ions.
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Reactivity of Thiol-Rich Zn Sites in Diacylglycerol-Sensing PKC C1 Domain Probed by NMR Spectroscopynull (Ed.)Conserved homology 1 (C1) domains are peripheral zinc finger domains that are responsible for recruiting their host signaling proteins, including Protein Kinase C (PKC) isoenzymes, to diacylglycerol-containing lipid membranes. In this work, we investigated the reactivity of the C1 structural zinc sites, using the cysteine-rich C1B regulatory region of the PKCα isoform as a paradigm. The choice of Cd 2+ as a probe was prompted by previous findings that xenobiotic metal ions modulate PKC activity. Using solution NMR and UV-vis spectroscopy, we found that Cd 2+ spontaneously replaced Zn 2+ in both structural sites of the C1B domain, with the formation of all-Cd and mixed Zn/Cd protein species. The Cd 2+ substitution for Zn 2+ preserved the C1B fold and function, as probed by its ability to interact with a potent tumor-promoting agent. Both Cys 3 His metal-ion sites of C1B have higher affinity to Cd 2+ than Zn 2+ , but are thermodynamically and kinetically inequivalent with respect to the metal ion replacement, despite the identical coordination spheres. We find that even in the presence of the oxygen-rich sites presented by the neighboring peripheral membrane-binding C2 domain, the thiol-rich sites can successfully compete for the available Cd 2+ . Our results indicate that Cd 2+ can target the entire membrane-binding regulatory region of PKCs, and that the competition between the thiol- and oxygen-rich sites will likely determine the activation pattern of PKCs.more » « less
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Pb 2+ is a xenobiotic metal ion that competes for Ca 2+ -binding sites in proteins. Using the peripheral Ca 2+ -sensing domains of Syt1, we show that the chelating pH buffer Bis–Tris enables identification and functional characterization of high-affinity Pb 2+ sites that are likely to be targeted by bioavailable Pb 2+ .more » « less