Graphene Nanoelectrodes: Fabrication and Size-Dependent Electrochemistry

The fabrication and electrochemistry of a new class of graphene electrodes are presented. Through high-temperature annealing of hydrazine-reduced graphene oxides followed by high-speed centrifugation and size-selected ultrafiltration, flakes of reduced graphene oxides (r-GOs) of nanometer and submicrometer dimensions, respectively, are obtained and separated from the larger ones. Using <i>n</i>-dodecanethiol-modified Au ultramicroelectrodes of appropriately small sizes, quick dipping in dilute suspensions of these small r-GOs allows attachment of only a single flake on the thiol monolayer. The electrodes thus fabricated are used to study the heterogeneous electron transfer (ET) kinetics at r-GOs and the nanoscopic charge transport dynamics at electrochemical interfaces. The r-GOs are found to exhibit similarly high activity for electrochemical ET reactions to metal electrodes. Voltammetric analysis for the relatively slow ET reaction of Fe­(CN)₆^3– reduction produces slightly higher ET rate constants at r-GOs of nanometer sizes than at large ones. These ET kinetic features are in accordance with the defect-dominant nature of the r-GOs and the increased defect density in the nanometer-sized flakes as revealed by Raman spectroscopic measurements. The voltammetric enhancement and inhibition for the reduction of Ru­(NH₃)₆³⁺ and Fe­(CN)₆^3–, respectively, at r-GO flakes of submicrometer and nanometer dimensions upon removal of supporting electrolyte are found to significantly deviate in magnitude from those predicted by the electroneutrality-based electromigration theory, which may evidence the increased penetration of the diffuse double layer into the mass transport layer at nanoscopic electrochemical interfaces

Characterization of the Zinc Uptake Repressor (Zur) from Acinetobacter baumannii

Bacterial cells tightly regulate the intracellular concentrations of essential transition metal ions by deploying a panel of metal-regulated transcriptional repressors and activators that bind to operator-promoter regions upstream of regulated genes. Like other zinc uptake regulator (Zur) proteins, Acinetobacter baumannii Zur represses transcription of its regulon when ZnII is replete and binds more weakly to DNA when ZnII is limiting. Previous studies established that Zur proteins are homodimeric and harbor at least two metal sites per protomer or four per dimer. CdII X-ray absorption spectroscopy (XAS) of the Cd2Zn2 AbZur metalloderivative with CdII bound to the allosteric sites reveals a S(N/O)3 first coordination shell. Site-directed mutagenesis suggests that H89 and C100 from the N-terminal DNA binding domain and H107 and E122 from the C-terminal dimerization domain comprise the regulatory metal site. KZn for this allosteric site is 6.0 (±2.2) × 1012 M–1 with a functional “division of labor” among the four metal ligands. N-terminal domain ligands H89 and C100 contribute far more to KZn than H107 and E122, while C100S AbZur uniquely fails to bind to DNA tightly as measured by an in vitro transcription assay. The heterotropic allosteric coupling free energy, ΔGc, is negative, consistent with a higher KZn for the AbZur-DNA complex and defining a bioavailable ZnII set-point of ≈6 × 10–14 M. Small-angle X-ray scattering (SAXS) experiments reveal that only the wild-type Zn homodimer undergoes allosteric switching, while the C100S AbZur fails to switch. These data collectively suggest that switching to a high affinity DNA-binding conformation involves a rotation/translation of one protomer relative to the other in a way that is dependent on the integrity of C100. We place these findings in the context of other Zur proteins and Fur family repressors more broadly

Characterization of the Zinc Uptake Repressor (Zur) from Acinetobacter baumannii

Bacterial cells tightly regulate the intracellular concentrations of essential transition metal ions by deploying a panel of metal-regulated transcriptional repressors and activators that bind to operator-promoter regions upstream of regulated genes. Like other zinc uptake regulator (Zur) proteins, Acinetobacter baumannii Zur represses transcription of its regulon when ZnII is replete and binds more weakly to DNA when ZnII is limiting. Previous studies established that Zur proteins are homodimeric and harbor at least two metal sites per protomer or four per dimer. CdII X-ray absorption spectroscopy (XAS) of the Cd2Zn2 AbZur metalloderivative with CdII bound to the allosteric sites reveals a S(N/O)3 first coordination shell. Site-directed mutagenesis suggests that H89 and C100 from the N-terminal DNA binding domain and H107 and E122 from the C-terminal dimerization domain comprise the regulatory metal site. KZn for this allosteric site is 6.0 (±2.2) × 1012 M–1 with a functional “division of labor” among the four metal ligands. N-terminal domain ligands H89 and C100 contribute far more to KZn than H107 and E122, while C100S AbZur uniquely fails to bind to DNA tightly as measured by an in vitro transcription assay. The heterotropic allosteric coupling free energy, ΔGc, is negative, consistent with a higher KZn for the AbZur-DNA complex and defining a bioavailable ZnII set-point of ≈6 × 10–14 M. Small-angle X-ray scattering (SAXS) experiments reveal that only the wild-type Zn homodimer undergoes allosteric switching, while the C100S AbZur fails to switch. These data collectively suggest that switching to a high affinity DNA-binding conformation involves a rotation/translation of one protomer relative to the other in a way that is dependent on the integrity of C100. We place these findings in the context of other Zur proteins and Fur family repressors more broadly

Exploiting GISAXS for the Study of a 3D Ordered Superlattice of Self-Assembled Colloidal Iron Oxide Nanocrystals

A three-dimensional (3D) ordered superlattice of colloidal iron oxide nanocrystals obtained by magnetic-field-assisted self-assembly has been studied by grazing incidence small-angle X-ray scattering (GISAXS). A new model to simulate and interpret GISAXS patterns is presented, which returns the structural and morphological details of 3D nanocrystal-built supercrystals. The model is applied to a sample with a suitable surface morphology, allowing the observation of “volume diffraction” even at extremely low grazing incidence angle. In this particular case, the average <i>fcc</i>-like stacking of the nanocrystals (building blocks), their spherical shape, and statistical information on their size distribution and positions within the superlattice have been safely deduced. The proposed model is expected to be amendable for the analysis of more complex structures and applicable to a large variety of nanocrystal-based assemblies

Structural and Functional Consequences of Three Cancer-Associated Mutations of the Oncogenic Phosphatase SHP2

The proto-oncogene <i>PTPN11</i> encodes a cytoplasmic protein tyrosine phosphatase, SHP2, which is required for normal development and sustained activation of the Ras-MAPK signaling pathway. Germline mutations in SHP2 cause developmental disorders, and somatic mutations have been identified in childhood and adult cancers and drive leukemia in mice. Despite our knowledge of the <i>PTPN11</i> variations associated with pathology, the structural and functional consequences of many disease-associated mutants remain poorly understood. Here, we combine X-ray crystallography, small-angle X-ray scattering, and biochemistry to elucidate structural and mechanistic features of three cancer-associated SHP2 variants harboring single point mutations within the N-SH2:PTP interdomain autoinhibitory interface. Our findings directly compare the impact of each mutation on autoinhibition of the phosphatase and advance the development of structure-guided and mutation-specific SHP2 therapies