No abstract

Nickel is a fundamental micronutrient for cellular life, but it is toxic in soluble form at nonphysiological concentrations. Such potentially contradictory features required living organisms to develop efficient systems for nickel utilization and homeostasis. This is the case for incorporation of nickel into the active site of urease, a multistep, tightly regulated process, requiring the interplay of various accessory proteins. The understanding of this activation mechanism may find medical applications against ureolytic bacteria, among which Mycobacterium tuberculosis is a deadly pathogen for humans. The topic of this study is UreG, an essential chaperone in the in vivo activation of urease upon insertion of Ni2+ into the active site. The protein was examined using both experimental and computational approaches. In particular, the soluble M. tuberculosis UreG (MtUreG) was overexpressed in Escherichia coli and purified to homogeneity. The identity of the isolated protein was established by mass spectrometry. On-line size-exclusion chromatography and light scattering indicated that MtUreG exists as a dimeric form in solution. Determination of the free thiol concentration revealed that a disulfide bond is present in the dimer. The isolated MtUreG shows low GTPase activity under native conditions, with a kcat of 0.01 min-1. Circular dichroism spectroscopy demonstrated the presence of a well-defined secondary structure (8% alpha-helices, 29% beta-strands) in MtUreG, whereas NMR spectroscopy indicated that this protein does not behave as a rigid three-dimensional fold and thus can be assigned to the class of intrinsically unstructured polypeptides. The molecular model of MtUreG in the fully folded and functional form was built using fold recognition algorithms. An extensive similarity search was performed to determine conservation patterns in all known bacterial UreG sequences. The generation of a multiple-sequence alignment and the related phylogenetic tree allowed us to recognize key residues and motifs that are likely important for protein function. A structural database containing the homology-built models of the most representative UreG proteins was created, confirming the structural analogies among the UreG family. A flexible region, likely to be important for protein function, is identified. The structural conservation among this class of GTPases is discussed on the basis of their function in the urease assembly process.

Force‐field and quantum‐chemical calculations are combined to model the packing of pentacene molecules at the atomic level on two polymer dielectric layers (poly(methyl methacrylate) (PMMA) versus polystyrene (PS)) widely used in field‐effect transistors and to assess the impact of electrostatic interactions at the interface on the charge mobility values in the pentacene layers. The results show unambiguously that the electrostatic interactions introduce a significant energetic disorder in the pentacene layer in contact with the polymer chains; a drop in the hole mobility by a factor of 5 is predicted with PS chains while a factor of 60 is obtained for PMMA due to the presence of polar carbonyl groups.