Publications by authors named "Emilia Chiancone"

50 Publications

Study of manganese binding to the ferroxidase centre of human H-type ferritin.

J Inorg Biochem 2018 05 9;182:103-112. Epub 2018 Feb 9.

INSTM, Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019 Sesto Fiorentino, Florence, Italy. Electronic address:

Ferritins are ubiquitous and conserved proteins endowed with enzymatic ferroxidase activity, that oxidize Fe(II) ions at the dimetal ferroxidase centre to form a mineralized Fe(III) oxide core deposited within the apo-protein shell. Herein, the in vitro formation of a heterodimetal cofactor constituted by Fe and Mn ions has been investigated in human H ferritin (hHFt). Namely, Mn and Fe binding at the hHFt ferroxidase centre and its effects on Fe(II) oxidation have been investigated by UV-Vis ferroxidation kinetics, fluorimetric titrations, multifrequency EPR, and preliminary Mössbauer spectroscopy. Our results show that in hHFt, both Fe(II) and Mn(II) bind the ferroxidase centre forming a Fe-Mn cofactor. Moreover, molecular oxygen seems to favour Mn(II) binding and increases the ferroxidation activity of the Mn-loaded protein. The data suggest that Mn influences the Fe binding and the efficiency of the ferroxidation reaction. The higher efficiency of the Mn-Fe heterometallic centre may have a physiological relevance in specific cell types (i.e. glia cells), where the concentration of Mn is the same order of magnitude as iron.
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http://dx.doi.org/10.1016/j.jinorgbio.2018.02.003DOI Listing
May 2018

A novel antimicrobial approach based on the inhibition of zinc uptake in Salmonella enterica.

Future Med Chem 2017 06 21;9(9):899-910. Epub 2017 Jun 21.

Consiglio Nazionale delle Ricerche (CNR)-Istituto di Biologia e Patologia Molecolari (IBPM) c/o Dipartimento di Scienze Biochimiche, Università Sapienza di Roma, P.le Aldo Moro 5, 00185 Rome, Italy.

In this review we discuss evidences suggesting that bacterial zinc homeostasis represents a promising target for new antimicrobial strategies. The ability of the gut pathogen Salmonella enterica sv Typhimurium to withstand the host responses aimed at controlling growth of the pathogen critically depends on the zinc importer ZnuABC. Strains lacking a functional ZnuABC or its soluble component ZnuA display a dramatic loss of pathogenicity, due to a reduced ability to express virulence factors; withstand the inflammatory response; and compete with other gut microbes. Based on this data, ZnuA was chosen as a candidate for the rational design of novel antibiotics. Through a combination of structural and functional investigations, we have provided a proof of concept of the potential of this approach.
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http://dx.doi.org/10.4155/fmc-2017-0042DOI Listing
June 2017

Salmonella enterica serovar Typhimurium growth is inhibited by the concomitant binding of Zn(II) and a pyrrolyl-hydroxamate to ZnuA, the soluble component of the ZnuABC transporter.

Biochim Biophys Acta 2016 Mar 12;1860(3):534-41. Epub 2015 Dec 12.

Department of Biochemical Sciences 'Sapienza' University of Rome, Piazzale A. Moro, 5, 00185 Rome, Italy. Electronic address:

Background: Under conditions of Zn(II) deficiency, the most relevant high affinity Zn(II) transport system synthesized by many Gram-negative bacteria is the ZnuABC transporter. ZnuABC is absent in eukaryotes and plays an important role in bacterial virulence. Consequently, ZnuA, the periplasmic component of the transporter, appeared as a good target candidate to find new compounds able to contrast bacterial growth by interfering with Zn(II) uptake.

Methods: Antibacterial activity assays on selected compounds from and in-house library against Salmonella enterica serovar Typhimurium ATCC14028 were performed. The X-ray structure of the complex formed by SeZnuA with an active compound was solved at 2.15Å resolution.

Results: Two di-aryl pyrrole hydroxamic acids differing in the position of a chloride ion, RDS50 ([1-[(4-chlorophenyl)methyl]-4-phenyl-1H-pyrrol-3-hydroxamic acid]) and RDS51 (1-[(2-chlorophenyl)methyl]-4-phenyl-1H-pyrrol-3-hydroxamic acid) were able to inhibit Salmonella growth and its invasion ability of Caco-2 cells. The X-ray structure of SeZnuA containing RDS51 revealed its presence at the metal binding site concomitantly with Zn(II) which is coordinated by protein residues and the hydroxamate moiety of the compound.

Conclusions: Two molecules interfering with ZnuA-mediated Zn(II) transport in Salmonella have been identified for the first time. The resolution of the SeZnuA-RDS51 X-ray structure revealed that RDS51 is tightly bound both to the protein and to Zn(II) thereby inhibiting its release. These features pave the way to the rational design of new Zn(II)-binding drugs against Salmonella.

General Significance: The data reported show that targeting the bacterial ZnuABC transporter can represent a good strategy to find new antibiotics against Gram-negative bacteria.
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http://dx.doi.org/10.1016/j.bbagen.2015.12.006DOI Listing
March 2016

Protecting Gram-negative bacterial cell envelopes from human lysozyme: Interactions with Ivy inhibitor proteins from Escherichia coli and Pseudomonas aeruginosa.

Biochim Biophys Acta 2015 Nov 31;1848(11 Pt B):3032-46. Epub 2015 Mar 31.

Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada. Electronic address:

Lysozymes play an important role in host defense by degrading peptidoglycan in the cell envelopes of pathogenic bacteria. Several Gram-negative bacteria can evade this mechanism by producing periplasmic proteins that inhibit the enzymatic activity of lysozyme. The Escherichia coli inhibitor of vertebrate lysozyme, Ivyc and its Pseudomonas aeruginosa homolog, Ivyp1 have been shown to be potent inhibitors of hen egg white lysozyme (HEWL). Since human lysozyme (HL) plays an important role in the innate immune response, we have examined the binding of HL to Ivyc and Ivyp1. Our results show that Ivyp1 is a weaker inhibitor of HL than Ivyc even though they inhibit HEWL with similar potency. Calorimetry experiments confirm that Ivyp1 interacts more weakly with HL than HEWL. Analytical ultracentrifugation studies revealed that Ivyp1 in solution is a monomer and forms a 30kDa heterodimer with both HL and HEWL, while Ivyc is a homodimer that forms a tetramer with both enzymes. The interaction of Ivyp1 with HL was further characterized by NMR chemical shift perturbation experiments. In addition to the characteristic His-containing Ivy inhibitory loop that binds into the active site of lysozyme, an extended loop (P2) between the final two beta-strands also participates in forming protein-protein interactions. The P2 loop is not conserved in Ivyc and it constitutes a flexible region in Ivyp1 that becomes more rigid in the complex with HL. We conclude that differences in the electrostatic interactions at the binding interface between Ivy inhibitors and distinct lysozymes determine the strength of this interaction. This article is part of a Special Issue entitled: Bacterial Resistance to Antimicrobial Peptides.
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http://dx.doi.org/10.1016/j.bbamem.2015.03.024DOI Listing
November 2015

The Salmonella enterica ZinT structure, zinc affinity and interaction with the high-affinity uptake protein ZnuA provide insight into the management of periplasmic zinc.

Biochim Biophys Acta 2014 Jan 12;1840(1):535-44. Epub 2013 Oct 12.

CNR Institute of Molecular Biology and Pathology and Department of Biochemical Sciences, "Sapienza" University of Rome, Piazzale A. Moro 5, 00185 Rome, Italy.

Background: In Gram-negative bacteria the ZnuABC transporter ensures adequate zinc import in Zn(II)-poor environments, like those encountered by pathogens within the infected host. Recently, the metal-binding protein ZinT was suggested to operate as an accessory component of ZnuABC in periplasmic zinc recruitment. Since ZinT is known to form a ZinT-ZnuA complex in the presence of Zn(II) it was proposed to transfer Zn(II) to ZnuA. The present work was undertaken to test this claim.

Methods: ZinT and its structural relationship with ZnuA have been characterized by multiple biophysical techniques (X-ray crystallography, SAXS, analytical ultracentrifugation, fluorescence spectroscopy).

Results: The metal-free and metal-bound crystal structures of Salmonella enterica ZinT show one Zn(II) binding site and limited structural changes upon metal removal. Spectroscopic titrations with Zn(II) yield a KD value of 22±2nM for ZinT, while those with ZnuA point to one high affinity (KD<20nM) and one low affinity Zn(II) binding site (KD in the micromolar range). Sedimentation velocity experiments established that Zn(II)-bound ZinT interacts with ZnuA, whereas apo-ZinT does not. The model of the ZinT-ZnuA complex derived from small angle X-ray scattering experiments points to a disposition that favors metal transfer as the metal binding cavities of the two proteins face each other.

Conclusions: ZinT acts as a Zn(II)-buffering protein that delivers Zn(II) to ZnuA.

General Significance: Knowledge of the ZinT-ZnuA relationship is crucial for understanding bacterial Zn(II) uptake.
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http://dx.doi.org/10.1016/j.bbagen.2013.10.010DOI Listing
January 2014

Kineococcus radiotolerans Dps forms a heteronuclear Mn-Fe ferroxidase center that may explain the Mn-dependent protection against oxidative stress.

Biochim Biophys Acta 2013 Jun 8;1830(6):3745-55. Epub 2013 Feb 8.

Department of Biochemical Sciences A. Rossi Fanelli, Sapienza University of Rome, Italy.

Background: The ferroxidase center of DNA-binding protein from starved cells (Dps) is a major player in the iron oxidation/detoxification process that leads to a decreased reactive oxygen species production. The possible Mn(II) participation in this process has been studied in Dps from Kineococcus radiotolerans, a radiation-resistant bacterium with a high cytosolic Mn/Fe ratio and a high capacity to survive ionizing and stress conditions.

Methods: The X-ray structure of recombinant K. radiotolerans Dps loaded with Mn(II) has been solved at 2.0Å resolution. Mn(II) binding to K. radiotolerans Dps and its effect on Fe(II) oxidation have been characterized in spectroscopic measurements.

Results: In K. radiotolerans Dps, the Fe-Fe ferroxidase center can have a Mn-Fe composition. Mn(II) binds only at the high affinity, so-called A site, whereas Fe(II) binds also at the low affinity, so-called B site. The Mn-Fe and Fe-Fe centers behave distinctly upon iron oxidation by O2. A site-bound Mn(II) or Fe(II) plays a catalytic role, while B site-bound Fe(II) behaves like a substrate and can be replaced by another Fe(II) after oxidation. When H2O2 is the Fe(II) oxidant, single electrons are transferred to aromatic residues near the ferroxidase center and give rise to intra-protein radicals thereby limiting OH release in solution. The presence of the Mn-Fe center results in significant differences in the development of such intra-protein radicals.

Conclusions: Mn(II) bound at the Dps ferroxidase center A site undergoes redox cycling provided the B site contains Fe.

General Significance: The results provide a likely molecular mechanism for the protective role of Mn(II) under oxidative stress conditions as it participates in redox cycling in the hetero-binuclear ferroxidase center.
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http://dx.doi.org/10.1016/j.bbagen.2013.02.003DOI Listing
June 2013

Effect of the charge distribution along the "ferritin-like" pores of the proteins from the Dps family on the iron incorporation process.

J Biol Inorg Chem 2011 Aug 6;16(6):869-80. Epub 2011 May 6.

Department of Biochemical Sciences, CNR Institute of Molecular Biology and Pathology, Sapienza University of Rome, Piazzale Aldo Moro, 5, 00185, Rome, Italy.

DNA-binding proteins from starved cells (Dps) differ in the number and position of charged residues along the "ferritin-like" pores that are used by iron to reach the ferroxidase center and the protein cavity. These differences are shown to affect significantly the electrostatic potential at the pores, which determines the extent of cooperativity in the iron uptake kinetics and thereby the mass distribution of the ferric hydroxide micelles inside the protein cavity. These conclusions are of biotechnological value in the preparation of protein-enclosed nanomaterials and are expected to apply also to ferritins. They were reached after characterization of the Dps from Listeria innocua, Helicobacter pylori, Thermosynechococcus elongatus, Escherichia coli, and Mycobacterium smegmatis. The characterization comprised the calculation of the electrostatic potential at the pores, determination of the iron uptake kinetics in the presence of molecular oxygen or hydrogen peroxide, and analysis of the proteins by means of the sedimentation velocity after iron incorporation.
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http://dx.doi.org/10.1007/s00775-011-0784-9DOI Listing
August 2011

The X-ray structure of the zinc transporter ZnuA from Salmonella enterica discloses a unique triad of zinc-coordinating histidines.

J Mol Biol 2011 Jun 21;409(4):630-41. Epub 2011 Apr 21.

CNR Institute of Molecular Biology and Pathology and Department of Biochemical Sciences, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy.

ZnuA is the soluble component of the high-affinity ZnuABC zinc transporter belonging to the cluster 9 group of ATP-binding cassette-type periplasmic Zn- and Mn-binding proteins. In Gram-negative bacteria, the ZnuABC system is essential for zinc uptake and homeostasis and is an important determinant of bacterial resistance to the host defense mechanisms. The cluster 9 members share a two (α/β)(4) domain architecture with a long α-helix connecting the two domains. In the Zn-specific proteins, the so-called α3c and the α4 helices are separated by an insert of variable length, rich in histidine and negatively charged residues. This distinctive His-rich loop is proposed to play a role in the management of zinc also due to its location at the entrance of the metal binding site located at the domain interface. The known Synechocystis 6803 and Escherichia coli ZnuA structures show the same metal coordination involving three conserved histidines and a glutamic acid or a water molecule as fourth ligand. The structures of Salmonella enterica ZnuA, with a partially or fully occupied zinc binding site, and of a deletion mutant missing a large part of the His-rich loop revealed unexpected differences in the metal-coordinating ligands, as histidine 140 from the mobile (at the C-terminal) part of the loop substitutes the conserved histidine 60. This unforeseen coordination is rendered possible by the "open conformation" of the two domains. The possible structural determinants of these peculiarities and their functional relevance are discussed.
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http://dx.doi.org/10.1016/j.jmb.2011.04.036DOI Listing
June 2011

The characterization of Thermotoga maritima ferritin reveals an unusual subunit dissociation behavior and efficient DNA protection from iron-mediated oxidative stress.

Extremophiles 2011 May 13;15(3):431-9. Epub 2011 Apr 13.

Institute of Molecular Biology & Pathology, CNR, Rome, Italy.

Ferritin from the hyperthermophilic anaerobe Thermotoga maritima, a bacterium of ancient phylogenetic origin, is structurally similar to known bacterial and eukaryotic ferritins: 24 identical subunits assemble into a shell having octahedral symmetry and a Mr of about 460 kDa. T. maritima ferritin (TmFtn), purified to homogeneity as a recombinant protein, contains approximately 2-3 iron atoms and can incorporate efficiently up to 3,500 atoms in the form of a ferric oxy-hydroxide mineral at 80°C, the optimal growth temperature of the bacterium. The 24-mer unexpectedly dissociates reversibly into dimers at low ionic strengths. In turn, dimers re-associate into the native 24-mer assembly at high protein concentrations and upon incorporation of iron micelles containing at least 500 Fe(III). TmFtn uses O(2) as efficient iron oxidant. The reaction stoichiometry is 3-4 O(2):Fe(II) as in all bacterial ferritins. Accordingly no H(2)O(2) is released into solution, a feature reflected in the in vitro ability of TmFtn to reduce significantly iron-mediated oxidative damage to DNA at 80°C. A similar TmFtn-mediated ROS detoxifying role likely occurs in the bacterium which lacks the SOD/catalase defense systems of the aerobic world.
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http://dx.doi.org/10.1007/s00792-011-0374-3DOI Listing
May 2011

Activation of the cardiac Na(+)-Ca(2+) exchanger by sorcin via the interaction of the respective Ca(2+)-binding domains.

J Mol Cell Cardiol 2010 Jul 15;49(1):132-41. Epub 2010 Mar 15.

C.N.R. Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi Fanelli, Sapienza University of Rome, 00185 Rome, Italy.

Sorcin is a penta-EF-hand protein that interacts with intracellular target proteins after Ca(2+) binding. The sarcolemmal Na(+)/Ca(2+) exchanger (NCX1) may be an important sorcin target in cardiac muscle. In this study, RNAi knockdown of sorcin, purified sorcin or sorcin variants was employed in parallel measurements of: (i) NCX activity in isolated rabbit cardiomyocytes using electrophysiological techniques and (ii) sorcin binding to the NCX1 calcium binding domains (CBD1 and (iii) using surface plasmon resonance and gel overlay techniques. Sorcin is activated by Ca(2+) binding to the EF3 and EF2 regions, which are connected by the D helix. To investigate the importance of this region in the interaction with NCX1, three variants were examined: W105G and W99G, mutated respectively near EF3 and EF2, and E124A that does not bind Ca(2+) due to a mutation at EF3. Downregulation of sorcin decreased and supplementation with wt sorcin (3muM) increased NCX activity in isolated cardiomyocytes. The relative stimulatory effects of the sorcin variants were: W105G>wt sorcin>Sorcin Calcium Binding Domain (SCBD)>W99G>E124A. Sorcin binding to both CBD1 and 2 was observed. In the presence of 50microM Ca(2+), the interaction with CBD1 followed the order W105G>SCBD>wt sorcin>W99G>E124A. In sorcin, the interacting surface can be mapped on the C-terminal Ca(2+)-binding domain in the D helix region comprising W99. The fast association/dissociation rates that characterize the interaction of sorcin with CBD1 and 2 may permit complex formation/dissociation during an excitation/contraction cycle.
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http://dx.doi.org/10.1016/j.yjmcc.2010.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2958309PMC
July 2010

The multifaceted capacity of Dps proteins to combat bacterial stress conditions: Detoxification of iron and hydrogen peroxide and DNA binding.

Biochim Biophys Acta 2010 Aug 4;1800(8):798-805. Epub 2010 Feb 4.

Department of Biochemical Sciences 'A. Rossi Fanelli', "Sapienza" University of Rome, Rome, Italy.

Background: The widely expressed Dps proteins, so named after the DNA-binding properties of the first characterized member of the family in Escherichia coli, are considered major players in the bacterial response to stress.

Scope Of Review: The review describes the distinctive features of the "ferritin-like" ferroxidation reaction, which uses hydrogen peroxide as physiological iron oxidant and therefore permits the concomitant removal of the two reactants that give rise to hydroxyl radicals via Fenton chemistry. It also illustrates the structural elements identified to date that render the interaction of some Dps proteins with DNA possible and outlines briefly the significance of Dps-DNA complex formation and of the Dps interaction with other DNA-binding proteins in relation to the organization of the nucleoid and microbial survival.

General Significance: Understanding in molecular terms the distinctive role of Dps proteins in bacterial resistance to general and specific stress conditions.

Major Conclusions: The state of the art is that the response to oxidative and peroxide-mediated stress is mediated directly by Dps proteins via their ferritin-like activity. In contrast, the response to other stress conditions derives from the concerted interplay of diverse interactions that Dps proteins may establish with DNA and with other DNA-binding proteins.
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http://dx.doi.org/10.1016/j.bbagen.2010.01.013DOI Listing
August 2010

Thermosynechococcus elongatus DpsA binds Zn(II) at a unique three histidine-containing ferroxidase center and utilizes O2 as iron oxidant with very high efficiency, unlike the typical Dps proteins.

FEBS J 2010 Feb 20;277(4):903-17. Epub 2010 Jan 20.

C.N.R. Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi-Fanelli, University of Rome La Sapienza, Italy.

The cyanobacterium Thermosynechococcus elongatus is one the few bacteria to possess two Dps proteins, DpsA-Te and Dps-Te. The present characterization of DpsA-Te reveals unusual structural and functional features that differentiate it from Dps-Te and the other known Dps proteins. Notably, two Zn(II) are bound at the ferroxidase center, owing to the unique substitution of a metal ligand at the A-site (His78 in place of the canonical aspartate) and to the presence of a histidine (His164) in place of a hydrophobic residue at a metal-coordinating distance in the B-site. Only the latter Zn(II) is displaced by incoming iron, such that Zn(II)-Fe(III) complexes are formed upon oxidation, as indicated by absorbance and atomic emission spectroscopy data. In contrast to the typical behavior of Dps proteins, where Fe(II) oxidation by H(2)O(2) is about 100-fold faster than by O(2), in DpsA-Te the ferroxidation efficiency of O(2) is very high and resembles that of H(2)O(2). Oxygraphic experiments show that two Fe(II) are required to reduce O(2), and that H(2)O(2) is not released into solution at the end of the reaction. On this basis, a reaction mechanism is proposed that also takes into account the formation of Zn(II)-Fe(III) complexes. The physiological significance of the DpsA-Te behavior is discussed in the framework of a possible localization of the protein at the thylakoid membranes, where photosynthesis takes place, with the consequent increased formation of reactive oxygen species.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07532.xDOI Listing
February 2010

Role of Dps (DNA-binding proteins from starved cells) aggregation on DNA.

Front Biosci (Landmark Ed) 2010 Jan 1;15:122-31. Epub 2010 Jan 1.

C.N.R. Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi-Fanelli, Sapienza, University of Rome, 00185 Rome, Italy.

The review outlines the experimental studies that have led to the current understanding at a molecular level of the protective role exerted by Dps proteins under stress conditions. After a brief description of the structural signatures and of the ferroxidase activity, which confers to all Dps proteins the capacity to decrease the hydroxyl radical induced DNA damage, the interaction of some family members with DNA is analysed. Special emphasis is given to the Dps structural elements that render the interaction with DNA possible and to the consequences that complex formation has on nucleoid organization and microbial survival.
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http://dx.doi.org/10.2741/3610DOI Listing
January 2010

Dps proteins prevent Fenton-mediated oxidative damage by trapping hydroxyl radicals within the protein shell.

Free Radic Biol Med 2010 Jan 3;48(2):292-7. Epub 2009 Nov 3.

CNR Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi-Fanelli, Sapienza University of Rome, 00185 Rome, Italy.

Dps (DNA-binding proteins from starved cells) proteins belong to a widespread bacterial family of proteins expressed under nutritional and oxidative stress conditions. In particular, Dps proteins protect DNA against Fenton-mediated oxidative stress, as they catalyze iron oxidation by hydrogen peroxide at highly conserved ferroxidase centers and thus reduce significantly hydroxyl radical production. This work investigates the possible generation of intraprotein radicals during the ferroxidation reaction by Escherichia coli and Listeria innocua Dps, two representative members of the family. Stopped-flow analyses show that the conserved tryptophan and tyrosine residues located near the metal binding/oxidation center are in a radical form after iron oxidation by hydrogen peroxide. DNA protection assays indicate that the presence of both residues is necessary to limit release of hydroxyl radicals in solution and the consequent oxidative damage to DNA. In general terms, the demonstration that conserved protein residues act as a trap that dissipates free electrons generated during the oxidative process brings out a novel role for the Dps protein cage.
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http://dx.doi.org/10.1016/j.freeradbiomed.2009.10.053DOI Listing
January 2010

Synthesis of iron oxide nanoparticles in Listeria innocua Dps (DNA-binding protein from starved cells): a study with the wild-type protein and a catalytic centre mutant.

Chemistry 2010 Jan;16(2):709-17

C.N.R. Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi-Fanelli, Sapienza, University of Rome, 00185 Rome, Italy.

A comparative analysis of the magnetic properties of iron oxide nanoparticles grown in the cavity of the DNA-binding protein from starved cells of the bacterium Listeria innocua, LiDps, and of its triple-mutant lacking the catalytic ferroxidase centre, LiDps-tm, is presented. TEM images and static and dynamic magnetic and electron magnetic resonance (EMR) measurements reveal that, under the applied preparation conditions, namely alkaline pH, high temperature (65 degrees C), exclusion of oxygen, and the presence of hydrogen peroxide, maghemite and/or magnetite nanoparticles with an average diameter of about 3 nm are mineralised inside the cavities of both LiDps and LiDps-tm. The magnetic nanoparticles (MNPs) thus formed show similar magnetic properties, with superparamagnetic behaviour above 4.5 K and a large magnetic anisotropy. Interestingly, in the EMR spectra an absorption at half-field is observed, which can be considered as a manifestation of the quantum behaviour of the MNPs. These results indicate that Dps proteins can be advantageously used for the production of nanomagnets at the interface between molecular clusters and traditional MNPs and that the presence of the ferroxidase centre, though increasing the efficiency of nanoparticle formation, does not affect the nature and fine structure of the MNPs. Importantly, the self-organisation of MNP-containing Dps on HRTEM grids suggests that Dps-enclosed MNPs can be deposited on surfaces in an ordered fashion.
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http://dx.doi.org/10.1002/chem.200901138DOI Listing
January 2010

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

Archaea 2009 Feb 17;2(4):221-31. Epub 2009 Feb 17.

Dipartimento di Scienze Biochimiche A.Rossi-Fanelli, Sapienza, Università di Roma, Roma, Italy.

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 degrees C to between 70 and 80 degrees 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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2686392PMC
http://dx.doi.org/10.1155/2009/280317DOI Listing
February 2009

Iron translocation into and out of Listeria innocua Dps and size distribution of the protein-enclosed nanomineral are modulated by the electrostatic gradient at the 3-fold "ferritin-like" pores.

J Biol Chem 2009 Jul 20;284(28):19101-9. Epub 2009 May 20.

CNR Institute of Molecular Biology and Pathology, Department of Biochemical Sciences, University of Rome, 00185 Rome, Italy.

Elucidating pore function at the 3-fold channels of 12-subunit, microbial Dps proteins is important in understanding their role in the management of iron/hydrogen peroxide. The Dps pores are called "ferritin-like" because of the structural resemblance to the 3-fold channels of 24-subunit ferritins used for iron entry and exit to and from the protein cage. In ferritins, negatively charged residues lining the pores generate a negative electrostatic gradient that guides iron ions toward the ferroxidase centers for catalysis with oxidant and destined for the mineralization cavity. To establish whether the set of three aspartate residues that line the pores in Listeria innocua Dps act in a similar fashion, D121N, D126N, D130N, and D121N/D126N/D130N proteins were produced; kinetics of iron uptake/release and the size distribution of the iron mineral in the protein cavity were compared. The results, discussed in the framework of crystal growth in a confined space, indicate that iron uses the hydrophilic 3-fold pores to traverse the protein shell. For the first time, the strength of the electrostatic potential is observed to modulate kinetic cooperativity in the iron uptake/release processes and accordingly the size distribution of the microcrystalline iron minerals in the Dps protein population.
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http://dx.doi.org/10.1074/jbc.M109.014670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2707210PMC
July 2009

Complex modulation of L-type Ca(2+) current inactivation by sorcin in isolated rabbit cardiomyocytes.

Pflugers Arch 2009 Mar 2;457(5):1049-60. Epub 2008 Sep 2.

University of Glasgow, UK.

Modulation of the L-type Ca(2+) channel (LTCC) by sorcin was investigated by measuring the L-type Ca(2+) current (I (Ca,L)) in isolated rabbit ventricular myocytes using ruptured patch, single electrode voltage clamp in the absence of extracellular Na(+). Fifty millimolars EGTA (170 nM Ca(2+)) in the pipette solution buffered bulk cytoplasmic [Ca(2+)], but retained rapid Ca(2+)-dependant inactivation of I (Ca,L,). Recombinant sorcin (3 microM) in the pipette significantly slowed time-dependant inactivation (tau (fast): 8.8 +/- 0.9 vs. 15.1 +/- 1.7 ms). Sorcin had no significant effect on I (Ca,L,) after inhibition of the sarcoplasmic reticulum (SR). Using 10 mM 1,2-bis(o-N,N,N',N'-tetraacetic acid (170 nM Ca(2+)), I (Ca,L) inactivation was then determined by a Ca(2+) -independent, voltage-dependant process. Under these conditions, 3 microM sorcin speeded up inactivation. A similar effect was observed by substitution of Ca(2+) with Ba(2+). Down-regulation of endogenous sorcin to 27 +/- 7% using an RNAi adenoviral vector slowed inactivation of I (Ca,L) by approximately 42%. The effects of sorcin on voltage-dependant inactivation were mimicked by a truncated form of the protein containing only the Ca(2+)-binding domain. This data is consistent with two independent actions of sorcin on the LTCC: (1) slowing Ca(2+)-dependant inactivation and (2) stimulating voltage-dependant inactivation. The net effect of sorcin on the time-dependent inactivation of I (Ca,L) was a balance between these two effects. Under normal conditions, sorcin slows I (Ca,L) inactivation because the effects of Ca(2+)-dependant inactivation out-weigh the effects on voltage-dependant inactivation.
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http://dx.doi.org/10.1007/s00424-008-0575-5DOI Listing
March 2009

The unusual co-assembly of H- and M-chains in the ferritin molecule from the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi.

Arch Biochem Biophys 2008 Oct 28;478(1):69-74. Epub 2008 Jun 28.

Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Sapienza, Università di Roma, P.le A. Moro, 5, 00185 Roma, Italy.

Ferritins from the liver and spleen of the cold-adapted Antarctic teleosts Trematomus bernacchii and Trematomus newnesi have been isolated and characterized. Interestingly, only H- and M-chains are expressed and no L-chains. The H-chains contain the conserved ferroxidase center residues while M-chains harbor both the ferroxidase center and the micelle nucleation site ligands. Ferritins have an organ-specific subunit composition, they are: M homopolymers in spleen and H/M heteropolymers in liver. The M-chain homopolymer mineralizes iron at higher rate with respect to the H/M heteropolymer, which however is endowed with a lower activation energy for the iron incorporation process, indicative of a higher local flexibility. These findings and available literature data on ferritin expression in fish point to the role of tissue-specific expression of different chains in modulating the iron oxidation/mineralization process.
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http://dx.doi.org/10.1016/j.abb.2008.06.022DOI Listing
October 2008

Sorcin modulates cardiac L-type Ca2+ current by functional interaction with the alpha1C subunit in rabbits.

Exp Physiol 2008 Dec 4;93(12):1233-8. Epub 2008 Jul 4.

Faculty of Biomedical & Life Sciences, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK.

We examined the modulation of the cardiac L-type Ca(2+) channel (LTCC) by the regulatory protein sorcin and tested the hypothesis that modulation occurred by direct interaction. Whole-cell patch-clamp recordings were made on native rabbit ventricular myocytes and HEK 293 cells expressing cardiac alpha(1C) subunits. In ventricular cells, sorcin increased peak current when using either Ca(2+) or Ba(2+) as charge carriers. In HEK 293 cells, sorcin increased peak current density when using Ba(2+) as a charge carrier but not when using Ca(2+). In ventricular myocytes, current inactivation (tau(fast), in ms) was slowed by sorcin with Ca(2+) as the charge carrier, whilst in the presence of Ba(2+) it was enhanced. In HEK 293 cells, sorcin significantly enhanced tau(fast), but no significant change was observed with Ba(2+). This trend was mimicked by the truncated peptide, sorcin Ca(2+)-binding domain, which lacks the N-terminal domain. These data suggest that sorcin interacts with LTCC via its C-terminal domain, which alters current magnitude and tau(fast). These effects appear to be influenced by the prevailing experimental conditions.
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http://dx.doi.org/10.1113/expphysiol.2008.043497DOI Listing
December 2008

Transcription of the Listeria monocytogenes fri gene is growth-phase dependent and is repressed directly by Fur, the ferric uptake regulator.

Gene 2008 Feb 28;410(1):113-21. Epub 2008 Jan 28.

Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università La Sapienza, Rome, Italy.

The Listeria monocytogenes fri gene encodes the only ferritin-like protein of this pathogen, a Dps protein (DNA binding protein from starved cells). Listeria Dps is endowed with the capacity to detoxify concurrently free iron and H(2)O(2), is essential for virulence and is required for efficient bacterial growth at early stages of the infection process. The transcription of fri is known to depend on sigma(A) and sigma(B) factors, to be affected by growth conditions and to be derepressed in a perR (peroxide-inducible stress response regulator) mutant background. The present work shows that fri transcription is restricted to the exponential phase of growth, whereas the Dps protein has a long half-life and is detected in significant amounts also in stationary phase cells. Expression of fri is downregulated under iron-rich conditions and is controlled directly by Fur, the ferric uptake regulator, which binds within the DNA region encompassing nucleotides from position -23 to position +90 relative to the proximal sigma(A) transcription startpoint. The putative Fur-box is proposed to coincide with the putative Per-box both in sequence and position. The primary structure of L. monocytogenes Fur has a high degree of similarity with homologues of known X-ray crystal structure. The molecular model of L. monocytogenes Fur built on this basis shows that the ligands of the structural Zn(II) and of the regulatory Fe(II) are conserved and are located in positions fully compatible with their respective roles.
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http://dx.doi.org/10.1016/j.gene.2007.12.007DOI Listing
February 2008

Periplasmic Cu,Zn superoxide dismutase and cytoplasmic Dps concur in protecting Salmonella enterica serovar Typhimurium from extracellular reactive oxygen species.

Biochim Biophys Acta 2008 Feb 14;1780(2):226-32. Epub 2007 Dec 14.

Dipartimento di Biologia, Universitá di Roma Tor Vergata, Via della Ricerca Scientifica, 00133 Roma, Italy.

Several bacteria possess periplasmic Cu,Zn superoxide dismutases which can confer protection from extracellular reactive oxygen species. Thus, deletion of the sodC1 gene reduces Salmonella enterica serovar Typhimurium ability to colonize the spleens of wild type mice, but enhances virulence in p47phox mutant mice. To look into the role of periplamic Cu,Zn superoxide dismutase and into possible additive effects of the ferritin-like Dps protein involved in hydrogen peroxide detoxification, we have analyzed bacterial survival in response to extracellular sources of superoxide and/or hydrogen peroxide. Exposure to extracellular superoxide of Salmonella Typhimurium mutant strains lacking the sodC1 and sodC2 genes and/or the dps gene does not cause direct killing of bacteria, indicating that extracellular superoxide is poorly bactericidal. In contrast, all mutant strains display a sharp hydrogen peroxide-dependent loss of viability, the dps,sodC1,sodC2 mutant being less resistant than the dps or the sodC1,sodC2 mutants. These findings suggest that the role of Cu,Zn superoxide dismutase in bacteria is to remove rapidly superoxide from the periplasm to prevent its reaction with other reactive molecules. Moreover, the nearly additive effect of the sodC and dps mutations suggests that localization of antioxidant enzymes in different cellular compartments is required for bacterial resistance to extracytoplasmic oxidative attack.
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http://dx.doi.org/10.1016/j.bbagen.2007.12.001DOI Listing
February 2008

Unusually strong H-bonding to the heme ligand and fast geminate recombination dynamics of the carbon monoxide complex of Bacillus subtilis truncated hemoglobin.

Biochemistry 2008 Jan 23;47(3):902-10. Epub 2007 Dec 23.

Dipartimento di Chimica, Università di Firenze, Via della Lastruccia 3, I-50019 Sesto Fiorentino (FI), Italy.

The active site of the oxygen-avid truncated hemoglobin from Bacillus subtilis has been characterized by infrared absorption and resonance Raman spectroscopies, and the dynamics of CO rebinding after photolysis has been investigated by picosecond transient absorption spectroscopy. Resonance Raman experiments on the CO bound adduct revealed the presence of two Fe-CO stretching bands at 545 and 520 cm-1, respectively. Accordingly, two C-O stretching bands at 1924 and 1888 cm-1 were observed in infrared absorption and resonance Raman measurements. The very low C-O stretching frequency at 1888 cm-1 (corresponding to the extremely high RR stretching frequency at 545 cm-1) indicates unusually strong hydrogen bonding between CO and distal residues. On the basis of a comparison with other truncated hemoglobin it is envisaged that the two CO conformers are determined by specific interactions with the TrpG8 and TyrB10 residues. Mutation of TrpG8 to Leu deeply alters the hydrogen-bonding network giving rise mainly to a CO conformer characterized by a Fe-CO stretching band at 489 cm-1 and a CO stretching band at 1958 cm-1. Picosecond laser photolysis experiments carried out on the CO bound adduct revealed dynamical processes that take place within a few nanoseconds after photolysis. Picosecond dynamics is largely dominated by CO geminate rebinding and is consistent with strong H-bonding contributions of TyrB10 and TrpG8 to ligand stabilization.
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http://dx.doi.org/10.1021/bi701297fDOI Listing
January 2008

Maurizio Brunori turns 70: nothing but a brilliant future behind him.

IUBMB Life 2007 Aug-Sep;59(8-9):475-6

Department of Biochemical Sciences, University of Roma La Sapienza, Roma, Italy.

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http://dx.doi.org/10.1080/15216540701291737DOI Listing
November 2007

Molecular basis for the impaired function of the natural F112L sorcin mutant: X-ray crystal structure, calcium affinity, and interaction with annexin VII and the ryanodine receptor.

FASEB J 2008 Jan 15;22(1):295-306. Epub 2007 Aug 15.

CNR Institute of Molecular Biology and Pathology, University Sapienza, P.le A.Moro 5, 00185 Rome, Italy.

The penta-EF hand protein sorcin participates in the modulation of Ca2+-induced calcium-release in the heart through the interaction with several Ca2+ channels such as the ryanodine receptor. The modulating activity is impaired in the recently described natural F112L mutant. The F112 residue is located at the end of the D helix next to Asp113, one of the calcium ligands in the EF3 hand endowed with the highest affinity for the metal. The F112L-sorcin X-ray crystal structure at 2.5 A resolution displays marked alterations in the EF3 hand, where the hydrogen bonding network established by Phe112 is disrupted, and in the EF1 region, which is tilted in both monomers that give rise to the dimer, the stable form of the molecule. In turn, the observed tilt is indicative of an increased flexibility of the N-terminal part of the molecule. The structural alterations result in a 6-fold decrease in calcium affinity with respect to the wild-type protein and to an even larger impairment of the interaction with annexin VII and of the ability of sorcin to interact with and inhibit ryanodine receptors. These results provide a plausible structural and functional framework that helps elucidate the phenotypic alterations of mice overexpressing F112L-sorcin.
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http://dx.doi.org/10.1096/fj.07-8988comDOI Listing
January 2008

The yeast penta-EF protein Pef1p is involved in cation-dependent budding and cell polarization.

Mol Microbiol 2007 Aug 19;65(4):1122-38. Epub 2007 Jul 19.

Istituto di Biologia e Patologia Molecolari, CNR, and Dipartemento di Genetica e Biologia Molecolare, Sapienza Università di Roma, P. le A. Moro 5, 00185 Rome, Italy.

Penta-EF-hand (PEF) proteins bind calcium and participate in a variety of calcium-dependent processes in vertebrates. In yeast, intracellular cations regulate processes like cell division and polarized growth. This study reports the identification of a unique PEF protein in Saccharomyces cerevisiae encoded by the uncharacterized open reading frame YGR058w. Pef1p has a long and unstructured N-terminal domain conserved in ascomycetes, and a highly conserved C-terminal calcium binding domain homologous to human ALG-2 and sorcin. Pef1p binds calcium and zinc and homodimerizes in vitro and in vivo like vertebrate homologues. Disruption of PEF1 induces defective growth in SDS and cation depletion conditions. Significantly, a critical substitution in the second EF hand (E218A) lowers the in vitro affinity for zinc and phenocopies growth defects. The dissection of protein-protein interactions and the cellular localization of Pef1p analogous to that of RAM pathway components controlling daughter-specific gene expression at the site of bud emergence bring out the importance of this novel protein. Our data suggest that cation homeostasis is involved in the control of polarized growth and in stress response in budding yeast.
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http://dx.doi.org/10.1111/j.1365-2958.2007.05852.xDOI Listing
August 2007

A novel chimera: the "truncated hemoglobin-antibiotic monooxygenase" from Streptomyces avermitilis.

Gene 2007 Aug 3;398(1-2):52-61. Epub 2007 May 3.

Department of Biochemical Sciences, University of Rome La Sapienza, P.le Aldo Moro 5, 00185 Rome, Italy.

Novel chimeric proteins made of a globin domain fused with a "cofactor free" monooxygenase domain have been identified within the Streptomyces avermitilis and Frankia sp. genomes by means of bioinformatics methods. Structure based sequence alignments show that the globin domains of both proteins can be unambiguously assigned to the truncated hemoglobin family, in view of the striking similarity to the truncated hemoglobins from Mycobacterium tuberculosis, Thermobifida fusca and Bacillus subtilis. In turn, the non-heme domains belong to a family of small (about 100 aminoacids) homodimeric proteins annotated as antibiotic biosynthesis monooxygenases, despite the lack of a cofactor (e.g., a metal, a flavin or a heme) necessary for oxygen activation. The chimeric protein from S. avermitilis has been cloned, expressed and characterized. The protein is a stable dimer in solution based on analytical ultracentrifugation experiments. The heme ligand binding properties with oxygen and carbonmonoxide resemble those of other Group II truncated hemoglobins. In addition, an oxygen dependent redox activity has been demonstrated towards easily oxidizable substrates such as menadiol and p-aminophenol. These findings suggest novel functional roles of truncated hemoglobins, which might represent a vast class of multipurpose oxygen activating/scavenging proteins whose catalytic action is mediated by the interaction with cofactor free monooxygenases.
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http://dx.doi.org/10.1016/j.gene.2007.01.038DOI Listing
August 2007

The neutrophil-activating Dps protein of Helicobacter pylori, HP-NAP, adopts a mechanism different from Escherichia coli Dps to bind and condense DNA.

Nucleic Acids Res 2007 19;35(7):2247-56. Epub 2007 Mar 19.

C.N.R. Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi-Fanelli, University of Rome La Sapienza, Rome, Italy.

The Helicobacter pylori neutrophil-activating protein (HP-NAP), a member of the Dps family, is a fundamental virulence factor involved in H.pylori-associated disease. Dps proteins protect bacterial DNA from oxidizing radicals generated by the Fenton reaction and also from various other damaging agents. DNA protection has a chemical component based on the highly conserved ferroxidase activity of Dps proteins, and a physical one based on the capacity of those Dps proteins that contain a positively charged N-terminus to bind and condense DNA. HP-NAP does not possess a positively charged N-terminus but, unlike the other members of the family, is characterized by a positively charged protein surface. To establish whether this distinctive property could be exploited to bind DNA, gel shift, fluorescence quenching and atomic force microscopy (AFM) experiments were performed over the pH range 6.5-8.5. HP-NAP does not self-aggregate in contrast to Escherichia coli Dps, but is able to bind and even condense DNA at slightly acid pH values. The DNA condensation capacity acts in concert with the ferritin-like activity and could be used to advantage by H.pylori to survive during host-infection and other stress challenges. A model for DNA binding/condensation is proposed that accounts for all the experimental observations.
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http://dx.doi.org/10.1093/nar/gkm077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1874666PMC
June 2007

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.

Proteins 2007 Mar;66(4):975-83

Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università La Sapienza, Rome, Italy.

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.
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http://dx.doi.org/10.1002/prot.21305DOI Listing
March 2007

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

Arch Biochem Biophys 2007 Jan 30;457(1):85-94. Epub 2006 Oct 30.

Department of Biochemical Sciences and CNR Institute of Molecular Biology and Pathology, University of Rome La Sapienza, P le Aldo Moro 5, 00185 Rome, Italy.

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.
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http://dx.doi.org/10.1016/j.abb.2006.09.033DOI Listing
January 2007