PubMed Health⌕ Search

Biomedical subjects

Emilia Chiancone

Publications and source records attributed to Emilia Chiancone.

At least 19 recordsLinked to original sources
PubMed2007

The mutations Lys 114 --> Gln and Asp 126 --> Asn disrupt an intersubunit salt bridge and convert Listeria innocua Dps into its natural mutant Listeria monocytogenes Dps. Effects on protein stability at Low pH.

The stability of the dodecameric Listeria monocytogenes Dps has been compared with that of the Listeria innocua protein. The two proteins differ only in two amino acid residues that form an intersubunit salt-bridge in L. innocua Dps. This salt-bridge is replaced by a hydrogen bonding network in L. monocytogenes Dps as revealed by the X-ray crystal structure. The resistance to low pH and high temperature was assayed for both Dps proteins under equilibrium conditions and kinetically. Despite the identical equilibrium behavior, significant differences in the kinetic stability and activation energy of the unfolding process are apparent at pH 1.5. The higher stability of L. monocytogenes Dps has been accounted for in terms of the persistence of the hydrogen bonding network at this low pH value. In contrast, the salt-bridge between Lys 114 and Asp 126 characteristic of L. innocua Dps is most likely abolished due to protonation of Asp 126.

Amino Acids↗
PubMed2006

Crystal structure and ligand binding properties of the truncated hemoglobin from Geobacillus stearothermophilus.

A novel truncated hemoglobin has been identified in the thermophilic bacterium Geobacillus stearothermophilus (Gs-trHb). The protein has been expressed in Escherichia coli, the 3D crystal structure (at 1.5 Angstroms resolution) and the ligand binding properties have been determined. The distal heme pocket displays an array of hydrogen bonding donors to the iron-bound ligands, including Tyr-B10 on one side of the heme pocket and Trp-G8 indole nitrogen on the opposite side. At variance with the highly similar Bacillus subtilis hemoglobin, Gs-trHb is dimeric both in the crystal and in solution and displays several unique structural properties. In the crystal cell, the iron-bound ligand is not homogeneously distributed within each distal site such that oxygen and an acetate anion can be resolved with relative occupancies of 50% each. Accordingly, equilibrium titrations of the oxygenated derivative in solution with acetate anion yield a partially saturated ferric acetate adduct. Moreover, the asymmetric unit contains two subunits and sedimentation velocity ultracentrifugation data confirm that the protein is dimeric.

Acetates↗
PubMed2006

The W105G and W99G sorcin mutants demonstrate the role of the D helix in the Ca(2+)-dependent interaction with annexin VII and the cardiac ryanodine receptor.

Sorcin, a 21.6 kDa two-domain penta-EF-hand (PEF) protein, when activated by Ca(2+) binding, interacts with target proteins in a largely uncharacterized process. The two physiological EF-hands EF3 and EF2 do not belong to a structural pair but are connected by the D helix. To establish whether this helix is instrumental in sorcin activation, two D helix residues were mutated: W105, located near EF3 and involved in a network of interactions, and W99, located near EF2 and facing solvent, were substituted with glycine. Neither mutation alters calcium affinity. The interaction of the W105G and W99G mutants with annexin VII and the cardiac ryanodine receptor (RyR2), requiring the sorcin N-terminal and C-terminal domain, respectively, was studied. Surface plasmon resonance experiments show that binding of annexin VII to W99G occurs at the same Ca(2+) concentration as that of the wild type, whereas W105G requires a significantly higher Ca(2+) concentration. Ca(2+) spark activity of isolated heart cells monitors the sorcin-RyR2 interaction and is unaltered by W105G but is reduced equally by W99G and the wild type. Thus, substitution of W105, via disruption of the network of D helix interactions, affects the capacity of sorcin to recognize and interact with either target at physiological Ca(2+) concentrations, while mutation of solvent-facing W99 has little effect. The D helix appears to amplify the localized structural changes that occur at EF3 upon Ca(2+) binding and thereby trigger a structural rearrangement that enables interaction of sorcin with its molecular targets. The same activation process may apply to other PEF proteins in view of the D helix conservation.

Amino Acid Sequence↗
PubMed2006

Antioxidant Dps protein from the thermophilic cyanobacterium Thermosynechococcus elongatus.

DNA-binding proteins from starved cells (Dps proteins) protect bacteria primarily from oxidative damage. They are composed of 12 identical subunits assembled with 23-symmetry to form a compact cage-like structure known to be stable at temperatures > 70 degrees C and over a wide pH range. Thermosynechococcus elongatus Dps thermostability is increased dramatically relative to mesophilic Dps proteins. Hydrophobic interactions at the dimeric and trimeric interfaces called Dps-like are replaced by salt bridges and hydrogen bonds, a common strategy in thermophiles. Moreover, the buried surface area at the least-extended Dps-like interface is significantly increased. A peculiarity of T. elongatus Dps is the presence of a chloride ion coordinated with threefold symmetry-related arginine residues lining the opening of the Dps-like pore toward the internal cavity. T. elongatus Dps conserves the unusual intersubunit ferroxidase centre that allows the Dps protein family to oxidize Fe(II) with hydrogen peroxide, thereby inhibiting free radical production via Fenton chemistry. This catalytic property is of special importance in T. elongatus (which lacks the catalase gene) in the protection of DNA and photosystems I and II from hydrogen peroxide-mediated oxidative damage.

Amino Acid Sequence↗
PubMed2006

The ferritin-like protein Frm is a target for the humoral immune response to Listeria monocytogenes and is required for efficient bacterial survival.

The identity and role of listerial antigens recognized by antibodies following listerial infection is largely unknown. We identified the listerial ferritin-like protein Frm as a target of the humoral response following infection of mice with pathogenic Listeria monocytogenes. Specific antibodies to Frm are detected in antisera of mice infected with a pathogenic strain but not in antisera of mice infected with a non-pathogenic L. innocua strain. Antibodies raised to purified listerial ferritin allowed us to demonstrate that the expression of Frm is both growth phase- and temperature-dependent. Using an isogenic Deltafrm mutant, we find that ferritin is essential for bacterial growth in chemically defined minimal media but not in complex media such as brain-heart infusion. Mutant bacteria also exhibit a defect in intracellular replication. The Deltafrm strain is hypersensitive to hydrogen peroxide indicating that the Frm is required for protection against reactive oxygen intermediates under various growth conditions. Animal studies show that Frm contributes to pathogenesis in mice, in particular, at early time points following infection.

Animals↗
PubMed2005

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

The structure and function of Mycobacterium smegmatis Dps (DNA-binding proteins from starved cells) and of the protein studied by Gupta and Chatterji, 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 pK(a) values of approximately 7.65 and 4.75; at pH approximately 4.0, dimers formed monomers. Based on structural analysis, the two dissociation steps have been attributed to breakage of the salt bridges between Glu(157) and Arg(99) located at the 3-fold symmetry axes and to protonation of Asp(66) hydrogen-bonded to Lys(36) 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.

Bacterial Proteins↗
PubMed2005

The so-called Listeria innocua ferritin is a Dps protein. Iron incorporation, detoxification, and DNA protection properties.

Listeria innocua Dps (DNA binding protein from starved cells) affords protection to DNA against oxidative damage and can accumulate about 500 iron atoms within its central cavity through a process facilitated by a ferroxidase center. The chemistry of iron binding and oxidation in Listeria Dps (LiDps, formerly described as a ferritin) using H(2)O(2) as oxidant was studied to further define the mechanism of iron deposition inside the protein and the role of LiDps in protecting DNA from oxidative damage. The relatively strong binding of 12 Fe(2+) to the apoprotein (K(D) approximately 0.023 microM) was demonstrated by isothermal titration calorimetry, fluorescence quenching, and pH stat experiments. Hydrogen peroxide was found to be a more efficient oxidant for the protein-bound Fe(2+) than O(2). Iron(II) oxidation by H(2)O(2) occurs with a stoichiometry of 2 Fe(2+)/H(2)O(2) in both the protein-based ferroxidation and subsequent mineralization reactions, indicating complete reduction of H(2)O(2) to H(2)O. Electron paramagnetic resonance (EPR) spin-trapping experiments demonstrated that LiDps attenuates the production of hydroxyl radical by Fenton chemistry. DNA cleavage assays showed that the protein, while not binding to DNA itself, protects it against the deleterious combination of Fe(2+) and H(2)O(2). The overall process of iron deposition and detoxification by LiDps is described by the following equations. For ferroxidation, Fe(2+) + Dps(Z)--> [(Fe(2+))-Dps](Z+1) + H(+) (Fe(2+) binding) and [(Fe(2+))-Dps](Z+1) + Fe(2+) + H(2)O(2) --> [(Fe(3+))(2)(O)(2)-Dps](Z+1) + 2H(+) (Fe(2+) oxidation/hydrolysis). For mineralization, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(O)OH((core)) + 4H(+) (Fe(2+) oxidation/hydrolysis). These reactions occur in place of undesirable odd-electron redox processes that produce hydroxyl radical.

Bacterial Proteins↗
PubMed2005

The unusual intersubunit ferroxidase center of Listeria innocua Dps is required for hydrogen peroxide detoxification but not for iron uptake. A study with site-specific mutants.

The role of the ferroxidase center in iron uptake and hydrogen peroxide detoxification was investigated in Listeria innocua Dps by substituting the iron ligands His31, His43, and Asp58 with glycine or alanine residues either individually or in combination. The X-ray crystal structures of the variants reveal only small alterations in the ferroxidase center region compared to the native protein. Quenching of the protein fluorescence was exploited to assess stoichiometry and affinity of metal binding. Substitution of either His31 or His43 decreases Fe(II) affinity significantly with respect to wt L. innocua Dps (K approximately 10(5) vs approximately 10(7) M(-)(1)) but does not alter the binding stoichiometry [12 Fe(II)/dodecamer]. In the H31G-H43G and H31G-H43G-D58A variants, binding of Fe(II) does not take place with measurable affinity. Oxidation of protein-bound Fe(II) increases the binding stoichiometry to 24 Fe(III)/dodecamer. However, the extent of fluorescence quenching upon Fe(III) binding decreases, and the end point near 24 Fe(III)/dodecamer becomes less distinct with increase in the number of mutated residues. In the presence of dioxygen, the mutations have little or no effect on the kinetics of iron uptake and in the formation of micelles inside the protein shell. In contrast, in the presence of hydrogen peroxide, with increase in the number of substitutions the rate of iron oxidation and the capacity to inhibit Fenton chemistry, thereby protecting DNA from oxidative damage, appear increasingly compromised, a further indication of the role of ferroxidation in conferring peroxide tolerance to the bacterium.

Amino Acid Substitution↗
PubMed2004

The truncated oxygen-avid hemoglobin from Bacillus subtilis: X-ray structure and ligand binding properties.

The group II truncated hemoglobin from Bacillus subtilis has been cloned, expressed, purified, and characterized. B. subtilis truncated hemoglobin is a monomeric protein endowed with an unusually high oxygen affinity (in the nanomolar range) such that the apparent thermodynamic binding constant for O2 exceeds that for CO by 1 order of magnitude. The kinetic basis of the high oxygen affinity resides mainly in the very slow rate of ligand release. The extremely stable ferrous oxygenated adduct is resistant to oxidation, which can be achieved only with oxidant in large excess, e.g. ferricyanide in 50-fold molar excess. The three-dimensional crystal structure of the cyano-Met derivative was determined at 2.15 A resolution. Although the overall fold resembles that of other truncated hemoglobins, the distal heme pocket displays a unique array of hydrophilic side chains in the topological positions that dominate the steric interaction with iron-bound ligands. In fact, the Tyr-B10, Thr-E7, and Gln-E11 oxygens on one side of the heme pocket and the Trp-G8 indole NE1 nitrogen on the other form a novel pattern of the "ligand-inclusive hydrogen bond network" described for mycobacterial HbO. On the proximal side, the histidine residue is in an unstrained conformation, and the iron-His bond is unusually short (1.91 A).

Amino Acid Sequence↗
PubMed2004

DNA condensation and self-aggregation of Escherichia coli Dps are coupled phenomena related to the properties of the N-terminus.

Escherichia coli Dps (DNA-binding proteins from starved cells) is the prototype of a DNA-protecting protein family expressed by bacteria under nutritional and oxidative stress. The role of the lysine-rich and highly mobile Dps N-terminus in DNA protection has been investigated by comparing the self-aggregation and DNA-condensation capacity of wild-type Dps and two N-terminal deletion mutants, DpsDelta8 and DpsDelta18, lacking two or all three lysine residues, respectively. Gel mobility and atomic force microscopy imaging showed that at pH 6.3, both wild type and DpsDelta8 self-aggregate, leading to formation of oligomers of variable size, and condense DNA with formation of large Dps-DNA complexes. Conversely, DpsDelta18 does not self-aggregate and binds DNA without causing condensation. At pH 8.2, DpsDelta8 and DpsDelta18 neither self-aggregate nor cause DNA condensation, a behavior also displayed by wild-type Dps at pH 8.7. Thus, Dps self-aggregation and Dps-driven DNA condensation are parallel phenomena that reflect the properties of the N-terminus. DNA protection against the toxic action of Fe(II) and H2O2 is not affected by the N-terminal deletions either in vitro or in vivo, in accordance with the different structural basis of this property.

Bacterial Proteins↗
PubMed2004

Iron and proteins for iron storage and detoxification.

Iron is required by most organisms, but is potentially toxic due to the low solubility of the stable oxidation state, Fe(III), and to the tendency to potentiate the production of reactive oxygen species, ROS. The reactivity of iron is counteracted by bacteria with the same strategies employed by the host, namely by sequestering the metal into ferritin, the ubiquitous iron storage protein. Ferritins are highly conserved, hollow spheres constructed from 24 subunits that are endowed with ferroxidase activity and can harbour up to 4500 iron atoms as oxy-hydroxide micelles. The release of the metal upon reduction can alter the microorganism-host iron balance and hence permit bacteria to overcome iron limitation. In bacteria, the relevance of the Dps (DNA-binding proteins from starved cells) family in iron storage-detoxification has been recognized recently. The seminal studies on the protein from Listeria innocua demonstrated that Dps proteins have ferritin-like activity and most importantly have the capacity to attenuate the production of ROS. This latter function allows bacterial pathogens that lack catalase, e.g. Porphyromonas gingivalis, to survive in an aerobic environment and resist to peroxide stress.

Bacterial Proteins↗
PubMed2003

Information transfer in the penta-EF-hand protein sorcin does not operate via the canonical structural/functional pairing. A study with site-specific mutants.

Sorcin is a typical penta-EF-hand protein that participates in Ca2+-regulated processes by translocating reversibly from cytosol to membranes, where it interacts with different target proteins in different tissues. Binding of two Ca2+/monomer triggers translocation, although EF1, EF2, and EF3 are potentially able to bind calcium at micromolar concentrations. To identify the functional pair, the conserved bidentate -Z glutamate in these EF-hands was mutated to yield E53Q-, E94A-, and E124A-sorcin, respectively. Limited structural perturbations occur only in E124A-sorcin due to involvement of Glu-124 in a network of interactions that comprise the long D helix connecting EF3 to EF2. The overall affinity for Ca2+ and for two sorcin targets, annexin VII and the ryanodine receptor, follows the order wild-type > E53Q- > E94A- > E124A-sorcin, indicating that disruption of EF3 has the largest functional impact and that disruption of EF2 and EF1 has progressively smaller effects. Based on this experimental evidence, EF3 and EF2, which are not paired in the canonical manner, are the functional EF-hands. Sorcin is proposed to be activated upon Ca2+ binding to EF3 and transmission of the conformational change at Glu-124 via the D helix to EF2 and from there to EF1 via the canonical structural/functional pairing. This mechanism may be applicable to all penta-EF-hand proteins.

Amino Acid Sequence↗
PubMed2003

The Dps protein of Agrobacterium tumefaciens does not bind to DNA but protects it toward oxidative cleavage: x-ray crystal structure, iron binding, and hydroxyl-radical scavenging properties.

Agrobacterium tumefaciens Dps (DNA-binding proteins from starved cells), encoded by the dps gene located on the circular chromosome of this plant pathogen, was cloned, and its structural and functional properties were determined in vitro. In Escherichia coli Dps, the family prototype, the DNA binding properties are thought to be associated with the presence of the lysine-containing N-terminal tail that extends from the protein surface into the solvent. The x-ray crystal structure of A. tumefaciens Dps shows that the positively charged N-terminal tail, which is 11 amino acids shorter than in the E. coli protein, is blocked onto the protein surface. This feature accounts for the lack of interaction with DNA. The intersubunit ferroxidase center characteristic of Dps proteins is conserved and confers to the A. tumefaciens protein a ferritin-like activity that manifests itself in the capacity to oxidize and incorporate iron in the internal cavity and to release it after reduction. In turn, sequestration of Fe(II) correlates with the capacity of A. tumefaciens Dps to reduce the production of hydroxyl radicals from H2O2 through Fenton chemistry. These data demonstrate conclusively that DNA protection from oxidative damage in vitro does not require formation of a Dps-DNA complex. In vivo, the hydroxyl radical scavenging activity of A. tumefaciens Dps may be envisaged to act in concert with catalase A to counteract the toxic effect of H2O2, the major component of the plant defense system when challenged by the bacterium.

Agrobacterium tumefaciens↗
PubMed2002

The expression of the dodecameric ferritin in Listeria spp. is induced by iron limitation and stationary growth phase.

The Gram-positive bacterium Listeria innocua possesses an authentic ferritin with an unusual dodecameric assemblage that resembles the quaternary structure of the DNA-binding proteins designated Dps (DNA-binding proteins from starved cells). The L. innocua gene encoding the above protein, termed ferritin from Listeria innocua (fri), has been localized on a 3-kb HindIII chromosomal fragment cloned in the Escherichia coli strain DH5alphaF'. DNA sequence analysis reveals an open reading frame of 468 nucleotides matching perfectly the amino acid sequence of the protein. Primer extension analysis indicates the presence of two transcriptional startpoints located 36 (proximal) and 85 nt (distal) upstream the fri start codon, respectively. Each transcriptional startpoint is preceded by suitably located -10 and -35 elements, which match the sigma(A) (proximal) and sigma(B) (distal) consensus sequences.In L. innocua and Liseria monocytogenes, fri expression increases both upon entry into stationary phase and, more markedly, under low-iron growth conditions. The effect of iron is apparent in the exponential and stationary phases of growth. An up-regulation by iron limitation has never been observed in other proven ferritins and bacterioferritins, but has been reported for several members of the Dps family. The unusual regulation by iron of the Listeria ferritin gene provides further support to the evolutionary link with the Dps family and suggests that the iron storage function may not be the unique role of ferritin in the physiology of this bacterium.

Amino Acid Sequence↗
PubMed2002

Iron incorporation into Escherichia coli Dps gives rise to a ferritin-like microcrystalline core.

Escherichia coli Dps belongs to a family of bacterial stress-induced proteins to protect DNA from oxidative damage. It shares with Listeria innocua ferritin several structural features, such as the quaternary assemblage and the presence of an unusual ferroxidase center. Indeed, it was recently recognized to be able to oxidize and incorporate iron. Since ferritins are endowed with the unique capacity to direct iron deposition toward formation of a microcrystalline core, the structure of iron deposited in the E. coli Dps cavity was studied. Polarized single crystal absorption microspectrophotometry of iron-loaded Dps shows that iron ions are oriented. The spectral properties in the high spin 3d(5) configuration point to a crystal form with tetrahedral symmetry where the tetrahedron center is occupied by iron ions and the vertices by oxygen. Crystals of iron-loaded Dps also show that, as in mammalian ferritins, iron does not remain bound to the site after oxidation has taken place. The kinetics of the iron reduction/release process induced by dithionite were measured in the crystal and in solution. The reaction appears to have two phases, with t(12) of a few seconds and several minutes at neutral pH values, as in canonical ferritins. This behavior is attributed to a similar composition of the iron core.

Bacterial Proteins↗
PubMed2002

The interplay between heme iron and protein sulfhydryls in the reaction of dimeric Scapharca inaequivalvis hemoglobin with nitric oxide.

The homodimeric hemoglobin from the mollusk Scapharca inaequivalvis possesses a single reactive cysteine residue per monomer, Cys92, which is located in the subunit interface in the vicinity of the heme group. The interplay between the heme iron and Cys92 towards the reaction with NO has been investigated by the combined use of electrospray mass spectrometry, FTIR and UV-Visible spectroscopy. When the ferrous liganded or unliganded protein reacts with free NO in solution Cys92 is not modified, but undergoes nitrosation when the hemoglobin is exposed to the nitric oxide releaser S-nitrosocysteine. When the ferric protein reacts with free NO under anaerobic conditions the heme iron is reduced and Cys92 is nitrosated. At variance with other hemeproteins investigated to date, in Scapharca HbI the heme-iron NO driven reduction is not accompanied by the formation of a ferric iron nitrosyl intermediate in detectable amounts. The results are consistent with the hypothesis that the nitrosating agent is the NO(+) species, which is generated during the NO driven reduction of the ferric heme iron. The possible reaction mechanism is discussed in comparison with recent findings on human hemoglobin and myoglobin.

Animals↗
PubMed2002

Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of Escherichia coli.

The DNA-binding proteins from starved cells (Dps) are a family of proteins induced in microorganisms by oxidative or nutritional stress. Escherichia coli Dps, a structural analog of the 12-subunit Listeria innocua ferritin, binds and protects DNA against oxidative damage mediated by H(2)O(2). Dps is shown to be a Fe-binding and storage protein where Fe(II) oxidation is most effectively accomplished by H(2)O(2) rather than by O(2) as in ferritins. Two Fe(2+) ions bind at each of the 12 putative dinuclear ferroxidase sites (P(Z)) in the protein according to the equation, 2Fe(2+) + P(Z) --> [(Fe(II)(2)-P](FS)(Z+2) + 2H(+). The ferroxidase site (FS) bound iron is then oxidized according to the equation, [(Fe(II)(2)-P](FS)(Z+2) + H(2)O(2) + H(2)O --> [Fe(III)(2)O(2)(OH)-P](FS)(Z-1) + 3H(+), where two Fe(II) are oxidized per H(2)O(2) reduced, thus avoiding hydroxyl radical production through Fenton chemistry. Dps acquires a ferric core of approximately 500 Fe(III) according to the mineralization equation, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(III)OOH((core)) + 4H(+), again with a 2 Fe(II)/H(2)O(2) stoichiometry. The protein forms a similar ferric core with O(2) as the oxidant, albeit at a slower rate. In the absence of H(2)O(2) and O(2), Dps forms a ferrous core of approximately 400 Fe(II) by the reaction Fe(2+) + H(2)O + Cl(-) --> Fe(II)OHCl((core)) + H(+). The ferrous core also undergoes oxidation with a stoichiometry of 2 Fe(II)/H(2)O(2). Spin trapping experiments demonstrate that Dps greatly attenuates hydroxyl radical production during Fe(II) oxidation by H(2)O(2). These results and in vitro DNA damage assays indicate that the protective effect of Dps on DNA most likely is exerted through a dual action, the physical association with DNA and the ability to nullify the toxic combination of Fe(II) and H(2)O(2). In the latter process a hydrous ferric oxide mineral core is produced within the protein, thus avoiding oxidative damage mediated by Fenton chemistry.

Bacterial Proteins↗