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The transcriptional regulator CueR is activated by the binding of CuI, AgI, or AuI to two cysteinates in a near-linear fashion. The C-terminal CCHHRAG sequence in Escherichia coli CueR present potential additional metal binding ligands and here we explore the effect of deleting this fragment on the binding of AgI to CueR. CD spectroscopic and ESI-MS data indicate that the high AgI-binding affinity of WT-CueR is significantly reduced in Δ7C-CueR.[111 Ag PAC spectroscopy demonstrates that the WT-CueR metal site structure (AgS2) is conserved, but less populated in the truncated variant. Thus, the function of the C-terminal fragment may be to stabilize the two-coordinate metal site for cognate monovalent metal ions. In a broader perspective this is an example of residues beyond the second coordination sphere affecting metal site physicochemical properties while leaving the structure unperturbed.

The response of CueR towards environmental changes in solution was investigated. CueR is a bacterial metal ion selective transcriptional metalloregulator protein, which controls the concentration of copper ions in the cell. Although several articles have been devoted to the discussion of the structural and functional features of this protein, CueR has not previously been extensively characterized in solution. Here, we studied the effect of change in pH, temperature, and the presence of specific or non-specific binding partners on the secondary structure of CueR with circular dichroism (CD) spectroscopy. A rather peculiar reversible pH-dependent secondary structure transformation was observed, elucidated and supplemented with pKa estimation by PROPKA and CpHMD simulations suggesting an important role of His(76) and His(94) in this process. CD experiments revealed that the presence of DNA prevents this structural switch, suggesting that DNA locks CueR in the α-helical-rich form. In contrast to the non-cognate metal ions HgII, CdII and ZnII, the presence of the cognate AgI ion affects the secondary structure of CueR, most probably by stabilizing the metal ion and DNA-binding domains of the protein.

Selectivity for monovalent metal ions is an important facet of the function of the metalloregulatory protein CueR. 111Ag perturbed angular correlation of γ‐rays (PAC) spectroscopy probes the metal site structure and the relaxation accompanying the instantaneous change from AgI to CdII upon 111Ag radioactive decay. That is, a change from AgI, which activates transcription, to CdII, which does not. In the frozen state (−196 °C) two nuclear quadrupole interactions (NQIs) are observed; one (NQI1) agrees well with two coordinating thiolates and an additional longer contact to the S77 backbone carbonyl, and the other (NQI2) reflects that CdII has attracted additional ligand(s). At 1 °C only NQI2 is observed, demonstrating that relaxation to this structure occurs within ≈10 ns of the decay of 111Ag. Thus, transformation from AgI to CdII rapidly disrupts the functional linear bis(thiolato)AgI metal site structure. This inherent metal site flexibility may be central to CueR function, leading to remodelling into a non‐functional structure upon binding of non‐cognate metal ions. In a broader perspective, 111Ag PAC spectroscopy may be applied to probe the flexibility of protein metal sites., Relax: AgI activates the transcriptional regulatory protein CueR, while CdII does not activate CueR. In this work the fact that 111Ag decays to 111Cd is exploited, to elucidate the subsequent relaxation at the metal site from transcriptional activator to repressor by PAC spectroscopy. Considerable metal site flexibility is observed, and it is hypothesized that this is relevant to the function of CueR.

199m Hg is a very useful probe for perturbed angular correlation of γ -rays (PAC) spectroscopy . Here we present 199m Hg PAC data for a Hg 2 + containing compound, Hg(Cys)2 (s), giving one unique nuclear quadrupole interaction (NQI). Four separate sample preparations were carried out, and measured on analogue and digital PAC setups, leading to the following reference data: ν Q = 1 . 458 ( 7 ) GHz and η = 0 . 10 ( 6 ) . The compound may be applied as an easy-to-synthesize reference sample, providing fast control of the effective anisotropy and time calibration of PAC instruments to be used for 199m Hg PAC experiments.

Intracellular CuI is controlled by the transcriptional regulator CueR, which effectively discriminates between monovalent and divalent metal ions. It is intriguing that HgII does not activate transcription, as bis‐thiolate metal sites exhibit high affinity for HgII. Here the binding of HgII to CueR and a truncated variant, ΔC7‐CueR, without the last 7 amino acids at the C‐terminus including a conserved CCHH motif is explored. ESI‐MS demonstrates that up to two HgII bind to CueR, while ΔC7‐CueR accommodates only one HgII. 199mHg PAC and UV absorption spectroscopy indicate HgS2 structure at both the functional and the CCHH metal site. However, at sub‐equimolar concentrations of HgII at pH 8.0, the metal binding site displays an equilibrium between HgS2 and HgS3, involving cysteines from both sites. We hypothesize that the C‐terminal CCHH motif provides auxiliary ligands that coordinate to HgII and thereby prevents activation of transcription., Intracellular CuI is controlled by the transcriptional regulator CueR, which effectively discriminates between monovalent and divalent metal ions. Here, the binding of HgII to CueR and a truncated variant, ΔC7‐CueR without the last seven amino acids at the C‐terminus including a conserved CCHH motif, is investigated.

We examined the response of algal cells to laboratory-induced cadmium stress in terms of physiological activity, autonomous features (motility and fluorescence), adhesion dynamics, nanomechanical properties, and protein expression by employing a multimethod approach. We develop a methodology based on the generalized mathematical model to predict free cadmium concentrations in culture. We used algal cells of Dunaliella tertiolecta, which are widespread in marine and freshwater systems, as a model organism. Cell adaptation to cadmium stress is manifested through cell shape deterioration, slower motility, and an increase of physiological activity. No significant change in growth dynamics showed how cells adapt to stress by increasing active surface area against toxic cadmium in the culture. It was accompanied by an increase in green fluorescence (most likely associated with cadmium vesicular transport and/or beta-carotene production), while no change was observed in the red endogenous fluorescence (associated with chlorophyll). To maintain the same rate of chlorophyll emission, the cell adaptation response was manifested through increased expression of the identified chlorophyll-binding protein(s) that are important for photosynthesis. Since production of these proteins represents cell defence mechanisms, they may also signal the presence of toxic metal in seawater. Protein expression affects the cell surface properties and, therefore, the dynamics of the adhesion process. Cells behave stiffer under stress with cadmium, and thus, the initial attachment and deformation are slower. Physicochemical and structural characterizations of algal cell surfaces are of key importance to interpret, rationalize, and predict the behaviour and fate of the cell under stress in vivo.

Intracellular CuI is controlled by the transcriptional regulator CueR, which effectively discriminates between monovalent and divalent metal ions. It is intriguing that HgII does not activate transcription, as bis-thiolate metal sites exhibit high affinity for HgII. Here the binding of HgII to CueR and a truncated variant, ΔC7-CueR, without the last 7 amino acids at the C-terminus including a conserved CCHH motif is explored. ESI-MS demonstrates that up to two HgII bind to CueR, while ΔC7-CueR accommodates only one HgII. 199mHg PAC and UV absorption spectroscopy indicate HgS2 structure at both the functional and the CCHH metal site. However, at sub-equimolar concentrations of HgII at pH 8.0, the metal binding site displays an equilibrium between HgS2 and HgS3, involving cysteines from both sites. We hypothesize that the C-terminal CCHH motif provides auxiliary ligands that coordinate to HgII and thereby prevents activation of transcription.

X-ray diffractometry dominates protein studies, as it can provide 3D structures of these diverse macromolecules or their molecular complexes with interacting partners: substrates, inhibitors, and/or cofactors. Here, we show that under cocrystallization conditions the results could reflect induced protein folds instead of the (partially) disordered original structures. The analysis of synchrotron radiation circular dichroism spectra revealed that the Im7 immunity protein stabilizes the native-like solution structure of unfolded NColE7 nuclease mutants via complex formation. This is consistent with the fact that among the several available crystal structures with its inhibitor or substrate, all NColE7 structures are virtually the same. Our results draw attention to the possible structural consequence of protein modifications, which is often hidden by compensational effects of intermolecular interactions. The growing evidence on the importance of protein intrinsic disorder thus, demands more extensive complementary experiments in solution phase with the unligated form of the protein of interest.

Metal ion regulation is essential for living organisms. In prokaryotes metal ion dependent transcriptional factors, the so-called metalloregulatory proteins play a fundamental role in controlling the concentration of metal ions. These proteins recognize metal ions with an outstanding selectivity. A detailed understanding of their function may be exploited in potential health, environmental and analytical applications. Members of the MerR protein family sense a broad range of mostly late transition and heavy metal ions through their cysteine thiolates. The air sensitivity of latter groups makes the expression and purification of such proteins challenging. Here we describe a method for the purification of the copper-regulatory CueR protein under optimized conditions. In order to avoid protein precipitation and/or eventual aggregation and to get rid of the co-purifying Escherichia coli elongation factor, our procedure consisted of four steps supplemented by DNA digestion. Subsequent anion exchange on Sepharose FF Q 16/10, affinity chromatography on Heparin FF 16/10, second anion exchange on Source 30 Q 16/13 and gel filtration on Superdex 75 26/60 resulted in large amounts of pure CueR protein without any affinity tag. Structure and functionality tests performed with mass spectrometry, circular dichroism spectroscopy and electrophoretic gel mobility shift assays approved the success of the purification procedure.

The enantioselective synthesis and electrochemistry of the first ferrocenyl GNA nucleosides is reported. These compounds were obtained by a Sharpless asymmetric dihydroxylation reaction of [3-(N1-thyminyl)-1-(ferrocenyl)]propene as S,R and R,S enantiomers in about 70 % yield with enantiomeric excesses of >99 % and 71 %, respectively. The absolute configurations of the chiral carbon atoms in the nucleosides were assigned by single-crystal X-ray diffraction analysis of the methyl derivatives in the solid state. The compounds were also studied with circular dichroism (CD) spectroscopy in solution. The enantiomeric relationship between the S,R and R,S isomers was confirmed by the near-mirror-image CD spectra. The redox properties of the nucleosides and their methylated derivatives were investigated using cyclic voltammetry. The cyclic voltammograms revealed reversible redox processes for the entire series of compounds at potentials of -25 mV (for nonmethylated derivatives) and 75 mV (for methylated derivatives) versus the ferrocene/ferrocenium reference redox couple.