Reassessment of protein stability, DNA binding, and protection of Mycobacterium smegmatis Dps.

Abstract The structure and function of Mycobacterium smegmatis Dps (DNA-binding proteins from starved cells) and of the protein studied by Gupta and Chatterji (Gupta, S., and Chatterji, D. (2003) J. Biol. Chem. 278, 5235-5241), in which the C terminus that is used for binding DNA contains a histidine tag, have been characterized in parallel. The native dodecamer dissociated reversibly into dimers above pH 7.5 and below pH 6.0, with apparent pKa values of ∼7.65 and 4.75; at pH ∼4.0, dimers formed monomers. Based on structural analysis, the two dissociation steps have been attributed to breakage of the salt bridges between Glu157 and Arg99 located at the 3-fold symmetry axes and to protonation of Asp66 hydrogen-bonded to Lys36 across the dimer interface, respectively. The C-terminal tag did not affect subunit dissociation, but altered DNA binding dramatically. At neutral pH, protonation of the histidine tag promoted DNA condensation, whereas in the native C terminus, compensation of negative and positive charges led to DNA binding without condensation. This different mode of interaction with DNA has important functional consequences as indicated by the failure of the native protein to protect DNA from DNase-mediated cleavage and by the efficiency of the tagged protein in doing so as a result of DNA sequestration in the condensates. Chemical protection of DNA from oxidative damage is realized by Dps proteins in a multistep iron oxidation/uptake/mineralization process. Dimers have a decreased protection efficiency due to disruption of the dodecamer internal cavity, where iron is deposited and mineralized after oxidation at the ferroxidase center

Hagfish Hemoglobins STRUCTURE, FUNCTION, AND OXYGEN-LINKED ASSOCIATION

Cyclostomes, hagfishes and lampreys, contain hemoglobins that are monomeric when oxygenated and polymerize to dimers or tetramers when deoxygenated. The three major hemoglobin components (HbI, HbII, and HbIII) from the hagfish Myxine glutinosa have been characterized and compared with lamprey Petromyzon marinus HbV, whose x-ray crystal structure has been solved in the deoxygenated, dimeric state (Heaslet, H. A., and Royer, W. E., Jr. (1999) Structure 7, 517-526). Of these three, HbII bears the highest sequence similarity to P. marinus HbV. In HbI and HbIII the distal histidine is substituted by a glutamine residue and additional substitutions occur in residues located at the deoxy dimer interface of P. marinus HbV. Infrared spectroscopy of the CO derivatives, used to probe the distal pocket fine structure, brings out a correlation between the CO stretching frequencies and the rates of CO combination. Ultracentrifugation studies show that HbI and HbIII are monomeric in both the oxygenated and deoxygenated states under all conditions studied, whereas deoxy HbII forms dimers at acidic pH values, like P. marinus HbV. Accordingly, the oxygen affinities of HbI and HbIII are independent of pH, whereas HbII displays a Bohr effect below pH 7.2. HbII also forms heterodimers with HbIII and heterotetramers with HbI. The functional counterparts of heteropolymer formation are cooperativity in oxygen binding and the oxygen-linked binding of protons and bicarbonate. The observed effects are explained on the basis of the x-ray structure of P. marinus HbV and the association behavior of site-specific mutants (Qiu, Y., Maillett, D. H., Knapp, J., Olson, J. S., and Riggs, A. F. (2000) J. Biol. Chem. 275, 13517-13528)

Semihemoglobins, High Oxygen Affinity Dimeric Forms of Human Hemoglobin Respond Efficiently to Allosteric Effectors without Forming Tetramers

Significant reduction in oxygen affinity resulting from interactions between heterotropic allosteric effectors and hemoglobin in not only the unligated derivative but also the fully ligated form has been reported (Tsuneshige, A., Park, S. I., and Yonetani, T. (2002) Biophys. Chem. 98, 49-63; Yonetani, T., Park, S. I., Tsuneshige, A., Imai, K., and Kanaori, K. (2002) J. Biol. Chem. 277, 34508-34520). To further investigate this effect in more detail, alpha- and beta-semihemoglobins, namely, alpha(heme)beta(apo) and alpha(apo)beta(heme), respectively, were prepared and characterized with respect to the impact of allosteric effectors on both conformation and ligand binding properties. Semihemoglobins are dimers characterized by a high affinity for oxygen and lack of cooperativity. We found that, compared with stripped conditions, semihemoglobins responded to effectors (inositol hexaphosphate and L35) by decreasing the affinity for oxygen by 60- and 130-fold for alpha- and beta-semihemoglobins, respectively. 1H NMR and sedimentation velocity experiments carried out with their ligated and unligated forms in the absence and presence of effectors revealed that semihemoglobins always remain as single-heme-carrying dimers. Recombination kinetics of their photolyzed CO derivatives showed that effectors did indeed interact with their ligated forms. Measurements of the Fe-His stretching mode show that the semihemoglobins undergo a large ligand binding-induced conformational shift and that both ligand-free and ligand derivatives respond to the presence of effectors. Contradictions to the Monod-Wyman-Changeaux/Perutz allosteric model arise since 1) the modulation of ligand affinity is not achieved in semihemoglobins by the formation of a low affinity T conformation (quaternary effect) but by direct interaction with effectors, 2) effectors do interact significantly with ligated forms of high affinity semihemoglobins, and 3) modulation of the ligand affinity and the cooperativity are not necessarily linked but instead can be separated into two distinct phenomena that can be isolated

Managing heat phenomena in epoxy composites production via graphenic derivatives: synthesis, properties and industrial production simulation of graphene and graphene oxide containing composites

A commercial two-components epoxy resin formulation was successfully modified by adding graphene and related materials (GRMs) and the effect of these nanofillers was assessed on their thermomechanical properties as well as on the simulation of their industrial application for the production of thick composites objects with interesting results. GMRs were added in different concentrations in order to improve thermo-mechanical properties of the nano-composite thermoset. Different dispersion methods were taken into account in order to produce stable long-lasting dispersion of the GRMs, that can withstand a commercial shelf life. Addition of the GRMs improves the glass transition temperature of the nanocomposite up to 20 °C with respect to the plain commercial formulation, and both stress and elongation at break increase up to almost 4 times the original values. Moreover, the industrial curing of some of the more promising modified resins was computer-simulated when the two-components resins are used to produce a carbon-fibre reinforced thick composite beam. Simulation results show that some of the applied GRMs helps reducing or even completely preventing the overheat phenomena that are well renown to induce significant thermal stresses negatively affecting the final object performances. These interesting effects would contribute reducing the time required for a single industrial production cycle, since no time for overheat dispersion is required, thus helping increasing the production rate

Oligomerization of Sulfolobus solfataricus

The recombinant amidase from the hyperthermophylic archaeon Sulfolobus solfataricus (SSAM) a signature amidase, was cloned, purified and characterized. The enzyme is active on a large number of aliphatic and aromatic amides over the temperature range 60–95 °C and at pH values between 4.0 and 9.5, with an optimum at pH 5.0. The recombinant enzyme is in the form of a dimer of about 110 kD that reversibly associates into an octamer in a pH-dependent reaction. The pH dependence of the state of association was studied using gel permeation chromatography, analytical ultracentrifugation and dynamic light scattering techniques

Multicentre Italian study of SARS-CoV-2 infection in children and adolescents, preliminary data as at 10 April 2020

Data on features of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in children and adolescents are scarce. We report preliminary results of an Italian multicentre study comprising 168 laboratory-confirmed paediatric cases (median: 2.3 years, range: 1 day-17.7 years, 55.9% males), of which 67.9% were hospitalised and 19.6% had comorbidities. Fever was the most common symptom, gastrointestinal manifestations were frequent; two children required intensive care, five had seizures, 49 received experimental treatments and all recovered

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes-7

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes is a series of editorials which is published on a biannual basis by the Editorial Board of the Medicinal Chemistry section of the journal Molecules. In these editorials, we highlight in brief reports (of about one hundred words) a number of recently published articles that describe crucial findings, such as the discovery of novel drug targets and mechanisms of action or novel classes of drugs, which may inspire future medicinal chemistry endeavors devoted to addressing prime unmet medical needs

pH-temperature and ion-dependent oligomerization of Sulfolobus solfataricus recombinant amidase:a study with site-specific mutants.

Recombinant amidase from Sulfolobus solfataricus occurred as a dimer of 110 kDa comprising identical subunits. Only dimers were present at pHs above 7.0, but with decreasing pH, dimers associated into octamers, with complete oligomerization occurring at pH 3.0. Oligomerization showed reversible temperature-dependence, with octamer formation increasing with temperature from 36 °C to between 70 and 80 °C. Increasing salt concentrations, favored dissociation of the octamers. Among the three investigated factors affecting the dimer–octamer equilibrium, the most important was pH. Among four mutants obtained by site-specific mutagenesis and selection for pH and temperature sensitivity, the T319I and D487N mutant amidases, like that of the native Sulfolobus solfataricus, responded to changes in pH and temperature with a conformational change affecting the dimer–octamer equilibrium. The Y41C and L34P mutant amidases were unaffected by pH and temperature, remaining always in the dimeric state. The differences among mutants in protein conformation must be related to the position of the introduced mutation. Although the L34P and Y41C mutations are located in the helical region 33–48 (LLKLQLESYERLDSLP), which is close to the amino-terminal segment of the protein, the T319I mutation is located in a strand on the surface of the protein, which is far from, and opposite to, the amino-terminal segment. The D487N mutation is located in the center of the protein, far distant from the 33–48 segment. These observations suggest that the segment of the protein closest to the amino-terminus plays a key role in the association of dimers into octamers