Co(II) electronic configuration allows its use as a spectroscopic probe in UV-Vis experiments to characterize the metal coordination sphere that is an essential component of the functional structure of zinc-binding proteins and to evaluate the metal ion affinities of these proteins. Here, exploiting the capability of the prokaryotic zinc finger to use different combinations of residues to properly coordinate the structural metal ion, we provide the UV-Vis characterization of Co(II) addition to Ros87 and its mutant Ros87_C27D which bears an unusual CysAspHis2 coordination sphere. Zinc finger sites containing only one cysteine have been infrequently characterized. We show for the CysAspHis2 coordination an intense d-d transition band, blue-shifted with respect to the Cys2His2 sphere. These data complemented by NMR and CD data demonstrate that the tetrahedral geometry of the metal site is retained also in the case of a single-cysteine coordination sphere.

Understanding the molecular determinants of fibrillogenesis by studying the aggregation propensities of high homologous proteins with different folding pathways.

Zinc ion binding is a principal event in the achievement of the correct fold in classical zinc finger domains since the motif is largely unfolded in the absence of metal. In the case of a prokaryotic zinc finger, the larger βββαα domain contributes to the folding mechanism with a larger hydrophobic core. For these reasons, following the great amount of attention devoted to unveiling the effect of xenobiotic metal ion replacement in zinc fingers and in zinc-containing proteins in general, the prokaryotic zinc finger domain appears to be an interesting model for studying metal ion interaction with metalloproteins. Here, we explore the binding of Ni­(II), Hg­(II), and Pb­(II) to Ros87, the DNA binding domain of the prokaryotic zinc finger protein Ros. We measured Ros87–metal ion dissociation constants and monitored the effects on the structure and function of the domain. Interestingly, we found that the protein folds in the presence of Ni­(II) with important structural perturbations, while in the presence of Pb­(II) and Hg­(II) it does not appear to be significantly folded. Accordingly, an overall strong reduction in the DNA binding capability is observed for all of the examined proteins. Our data integrate and complement the information collected in the past few years concerning the functional and structural effects of metal ion substitution in classical zinc fingers in order to contribute to a better comprehension of the toxicity of these metals in biological systems.

Enzyme I (EI) of the bacterial phosphotransferase system (PTS) is a master regulator of bacterial metabolism and a promising target for development of a new class of broad-spectrum antibiotics. The catalytic activity of EI is mediated by several intradomain, interdomain, and intersubunit conformational equilibria. Therefore, in addition to its relevance as a drug target, EI is also a good model for investigating the dynamics/function relationship in multidomain, oligomeric proteins. Here, we use solution NMR and protein design to investigate how the conformational dynamics occurring within the N-terminal domain (EIN) affect the activity of EI. We show that the rotameric g+-to-g− transition of the active site residue His189 χ2 angle is decoupled from the state A-to-state B transition that describes a ∼90° rigid-body rearrangement of the EIN subdomains upon transition of the full-length enzyme to its catalytically competent closed form. In addition, we engineered EIN constructs with modulated conformational dynamics by hybridizing EIN from mesophilic and thermophilic species, and used these chimeras to assess the effect of increased or decreased active site flexibility on the enzymatic activity of EI. Our results indicate that the rate of the autophosphorylation reaction catalyzed by EI is independent from the kinetics of the g+-to-g− rotameric transition that exposes the phosphorylation site on EIN to the incoming phosphoryl group. In addition, our work provides an example of how engineering of hybrid mesophilic/thermophilic chimeras can assist investigations of the dynamics/function relationship in proteins, therefore opening new possibilities in biophysics.

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