The 800-MHz 1H NMR spectra of oxidized plastocyanin from spinach are here reported. All hyperfine-shifted signals have been assigned through saturation transfer with the reduced diamagnetic species. To detect the copper(II)-bound cysteine β-CH2 signals, a technique has been applied which is based on irradiation of regions where such signals are expected but not detected, and the corresponding saturation transfer on the reduced species is observed. At the end, a full spectrum is reconstructed which permits, for the first time, the complete 1H NMR signal assignment of an oxidized blue copper protein. These data are discussed in terms of the factors affecting the line width as related to the electronic and geometric structure of the metal center. A Karplus-type relationship is proposed between the contact shift of the Cys-84 β-CH2 protons and the Cu−S−C−Hβ dihedral angle.

Bacillus pasteurii UreG, a chaperone involved in the urease active site assembly, was overexpressed in Escherichia coli BL21(DE3) and purified to homogeneity. The identity of the recombinant protein was confirmed by SDS-PAGE, protein sequencing, and mass spectrometry. A combination of size exclusion chromatography and multiangle and dynamic laser light scattering established that BpUreG is present in solution as a dimer. Analysis of circular dichroism spectra indicated that the protein contains large portions of helices (15%) and strands (29%), whereas NMR spectroscopy indicated the presence of conformational fluxionality of the protein backbone in solution. BpUreG catalyzes the hydrolysis of GTP with a k cat ‫؍‬ 0.04 min ؊1 , confirming a role for this class of proteins in coupling energy requirements and nickel incorporation into the urease active site. BpUreG binds two Zn 2؉ ions per dimer, with a K D ‫؍‬ 42 ؎ 3 M, and has a 10-fold lower affinity for Ni 2؉ . A structural model for BpUreG was calculated by using threading algorithms. The protein, in the fully folded state, features the typical structural architecture of GTPases, with an open ␤-barrel surrounded by ␣-helices and a P-loop at the N terminus. The protein dynamic behavior observed in solution is critically discussed relative to the structural model, using algorithms for disorder predictions. The results suggest that UreG proteins belong to the class of intrinsically unstructured proteins that need the interaction with cofactors or other protein partners to perform their function. It is also proposed that metal ions such as Zn 2؉ could have important structural roles in the urease activation process.

Selenium is an essential trace element in many life forms due to its occurrence as a selenocysteine (Sec) residue in selenoproteins. The majority of mammalian selenoproteins, however, have no known function. Herein, we performed extensive sequence similarity searches to define and characterize a new protein family, designated Rdx, that includes mammalian selenoproteins SelW, SelV, SelT and SelH, bacterial SelW-like proteins and cysteine-containing proteins of unknown function in all three domains of life. An additional member of this family is a mammalian cysteine-containing protein, designated Rdx12, and its fish selenoprotein orthologue. Rdx proteins are proposed to possess a thioredoxin-like fold and a conserved CxxC or CxxU (U is Sec) motif, suggesting a redox function. We cloned and characterized three mammalian members of this family, which showed distinct expression patterns in mouse tissues and different localization patterns in cells transfected with the corresponding GFP fusion proteins. By analogy to thioredoxin, Rdx proteins can use catalytic cysteine (or Sec) to form transient mixed disulfides with substrate proteins. We employed this property to identify cellular targets of Rdx proteins using affinity columns containing mutant versions of these proteins. Rdx12 was found to interact with glutathione peroxidase 1, whereas 14-3-3 protein was identified as one of the targets of mammalian SelW, suggesting a mechanism for redox regulation of the 14-3-3 family of proteins.