Dataset Information

Reversed Cation Selectivity of G₈ -Octamer and G₁₆ -Hexadecamer towards Monovalent and Divalent Cations.

ABSTRACT: A reverse-binding-selectivity between monovalent and divalent cations was observed for two different self-assembly G₁₆ -hexadecamer and G₈ -octamer systems. The dissociation constant between G₄ -quadruplex and monomer was calculated via VT-¹ H NMR experiments. Quantitative energy profiles revealed entropy as the key factor for the weaker binding toward Ba²⁺ compared with K⁺ in the G₈ -octamer system despite stronger ion-dipole interactions. This study is the first direct comparison of the G₄ -quartet binding affinity between mono and divalent cations and will benefit future applications of G-quadruplex-related research. Further competition experiments between the G₈ -octamer and 18-crown-6 with K⁺ demonstrated the potential of this G₈ system as a new potassium receptor.

SUBMITTER: He Y

PROVIDER: S-EPMC7138703 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Publications

Reversed Cation Selectivity of G8 -Octamer and G16 -Hexadecamer towards Monovalent and Divalent Cations.

He Ying Y Zhang Yanbin Y Wojtas Lukasz L Akhmedov Novruz G NG Pan Qinhe Q Guo Hao H Shi Xiaodong X

Chemistry, an Asian journal 20200306 7

A reverse-binding-selectivity between monovalent and divalent cations was observed for two different self-assembly G16 -hexadecamer and G8 -octamer systems. The dissociation constant between G4 -quadruplex and monomer was calculated via VT-1 H NMR experiments. Quantitative energy profiles revealed entropy as the key factor for the weaker binding toward Ba2+ compared with K+ in the G8 -octamer system despite stronger ion ...[more]

PMID: 32017419

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Project description:In voltage-gated ion channels, residues responsible for ion selectivity were identified in the pore-lining SS1-SS2 segments. Negatively charged glutamate residues (E393, E736, E1145, and E1446) found in each of the four repeats of the alpha 1C subunit were identified as the major determinant of selectivity in Ca2+ channels. Neutralization of glutamate residues by glutamine in repeat I (E393Q), repeat III (E1145Q), and repeat IV (E1446Q) decreased the channel affinity for calcium ions 10-fold from the wild-type channel. In contrast, neutralization of glutamate residues in repeat II failed to significantly alter Ca2+ affinity. Likewise, mutation of neighboring residues in E1149K and D1450N did not affect the channel affinity, further supporting the unique role of glutamate residues E1145 in repeat III and E1446 in repeat IV in determining Ca2+ selectivity. Conservative mutations E1145D and E1446D preserved high-affinity Ca2+ binding, which suggests that the interaction between Ca2+ and the pore ligand sites is predominantly electrostatic and involves charge neutralization. Mutational analysis of E1446 showed additionally that polar residues could achieve higher Ca2+ affinity than small hydrophobic residues could. The role of high-affinity calcium binding sites in channel permeation was investigated at the single-channel level. Neutralization of glutamate residue in repeats I, II, and III did not affect single-channel properties measured with 115 mM BaCl2. However, mutation of the high-affinity binding site E1446 was found to significantly affect the single-channel conductance for Ba2+ and Li+, providing strong evidence that E1446 is located in the narrow region of the channel outer mouth. Side-chain substitutions at 1446 in repeat IV were used to probe the nature of divalent cation-ligand interaction and monovalent cation-ligand interaction in the calcium channel pore. Monovalent permeation was found to be inversely proportional to the volume of the side chain at position 1446, with small neutral residues such as alanine and glycine producing higher Li+ currents than the wild-type channel. This suggests that steric hindrance is a major determinant for monovalent cation conductance. Divalent permeation was more complex. Ba2+ single-channel conductance decreased when small neutral residues such as glycine were replaced by bulkier ones such as glutamine. However, negatively charged amino acids produced single-channel conductance higher than predicted from the size of their side chain. Hence, negatively charged residues at position 1446 in repeat IV are required for divalent cation permeation.

Project description:We report on the influence of pH and monovalent/divalent cations on the catalytic current response, internal electron transfer (IET), and structure of fructose dehydrogenase (FDH) by using amperometry, spectrophotometry, and circular dichroism (CD). Amperometric measurements were performed on graphite electrodes, onto which FDH was adsorbed and the effect on the response current to fructose was investigated when varying the pH and the concentrations of divalent/monovalent cations in the contacting buffer. In the presence of 10 mM CaCl2, a current increase of up to ≈ 240% was observed, probably due to an intra-complexation reaction between Ca2+ and the aspartate/glutamate residues found at the interface between the dehydrogenase domain and the cytochrome domain of FDH. Contrary to CaCl2, addition of MgCl2 did not show any particular influence, whereas addition of monovalent cations (Na+ or K+) led to a slight linear increase in the maximum response current. To complement the amperometric investigations, spectrophotometric assays were carried out under homogeneous conditions in the presence of a 1-electron non-proton-acceptor, cytochrome c, or a 2-electron-proton acceptor, 2,6-dichloroindophenol (DCIP), respectively. In the case of cytochrome c, it was possible to observe a remarkable increase in the absorbance up to 200% when 10 mM CaCl2 was added. However, by further increasing the concentration of CaCl2 up to 50 mM and 100 mM, a decrease in the absorbance with a slight inhibition effect was observed for the highest CaCl2 concentration. Addition of MgCl2 or of the monovalent cations shows, surprisingly, no effect on the electron transfer to the electron acceptor. Contrary to the case of cytochrome c, with DCIP none of the cations tested seem to affect the rate of catalysis. In order to correlate the results obtained by amperometric and spectrophotometric measurements, CD experiments have been performed showing a great structural change of FDH when increasing the concentration CaCl2 up to 50 mM, at which the enzyme molecules start to agglomerate, hindering the substrate access to the active site probably due to a chelation reaction occurring at the enzyme surface with the glutamate/aspartate residues. Graphical Abstract Fructose dehydrogenase (FDH) consists of three subunits, but only two are involved in the electron transfer process: (I) 2e-/2H+ fructose oxidation, (II) internal electron transfer (IET), (III) direct electron transfer (DET) through 2 heme c; FDH activity either in solution or when immobilized onto an electrode surface is enhanced about 2.5-fold by adding 10 mM CaCl2 to the buffer solution, whereas MgCl2 had an "inhibition" effect. Moreover, the additions of KCl or NaCl led to a slight current increase.

Dataset Information

Reversed Cation Selectivity of G₈ -Octamer and G₁₆ -Hexadecamer towards Monovalent and Divalent Cations.

Publications

Reversed Cation Selectivity of G<sub>8</sub> -Octamer and G<sub>16</sub> -Hexadecamer towards Monovalent and Divalent Cations.

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