Algal cell response to laboratory‑induced cadmium stress: a multimethod approach

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

S77C-ΔC7-CueR: a 199mHg PAC study of the protein metal site structure

The CueR protein regulates the cytosolic concentration of Cu(I) in bacteria such as E. coli. With this work we aimed to remodel the linear two-coordinate metal site with Cys112 and Cys120 as ligands in CueR to a tricoordinate site similar to that observed in the Hg(II) sensor protein MerR. This was done by introducing an additional cysteine near the metal site in the modified S77C-ΔC7-CueR variant, inspired by the fact that Ser77 in CueR is replaced by a cysteine in MerR. 199m Hg PAC spectroscopic data indicate that two NQIs are present at pH 8.0, most likely reflecting HgS 2 and HgS 3 coordination modes, and demonstrating that the design of a pure HgS 3 metal site was not achieved. Lowering the pH to 6.0 or the temperature to −196 °C had surprisingly similar effects, giving rise to highly distorted trigonal Hg(II) coordination. Tentatively, this might reflect that the histidine just next to Cys77 (His76) coordinates forming a HgS 2 N metal site structure. Further redesign beyond the first coordination sphere appears to be required to efficiently stabilize the HgS 3 metal site structure at physiological pH

C‐terminal Cysteines of CueR Act as Auxiliary Metal Site Ligands upon Hg II Binding—A Mechanism To Prevent Transcriptional Activation by Divalent Metal Ions?

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. $^{199}$mHg PAC and UV absorption spectroscopy indicate HgS$_2$ 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 HgS$_2$ and HgS$_3$ , 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

Flexibility of the CueR metal site probed by instantaneous change of element and oxidation state from AgI to CdII

Selectivity for monovalent metal ions is an important facet of the function of the metalloregulatory protein CueR. 111Ag perturbed angular correlation of g-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 8C) 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 8C 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 re-modelling 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