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Ivano Bertini

Publications and source records attributed to Ivano Bertini.

At least 73 records · Page 4Linked to original sources
PubMed2003

The magnetic properties of myoglobin as studied by NMR spectroscopy.

Deoxymyoglobin has been investigated by NMR spectroscopy to determine the magnetic anisotropy through pseudocontact shifts and the total magnetic susceptibility through Evans measurements. The magnetic anisotropy values were found to be Deltachi(ax)=-2.03+/-0.08 x 10(-32) m(3) and Deltachi(rh)=-1.02+/-0.09 x 10(-32) m(3). The negative value of the axial susceptibility anisotropy originates from the z tensor axis lying in the heme plane, unlike all other heme systems investigated so far. This magnetic axis is almost exactly orthogonal to the axial histidine plane. The other two axes lie essentially in the histidine plane, the closest to the heme normal being tilted by about 36 degrees from it, towards pyrrole A on the side of the proximal histidine. From the comparison with cytochrome c' it clearly appears that the position of the one axis lying in the heme plane is related to the axial histidine orientation. Irrespective of the directions, the magnetic anisotropy is smaller than that of the analogous reduced cytochrome c' and of the order of that of low-spin iron(III). The magnetic anisotropy of the system permits the measurement of residual dipolar couplings, which, together with pseudocontact shifts, prove that the solution structure is very similar to that in the crystalline state. Magnetic measurements, at variance with previous data, demonstrate that there is an orbital contribution to the magnetic moment, micro(eff)=5.5 micro(B). Finally, from the magnetic anisotropy data, the hyperfine shifts of iron ligands could be separated in pseudocontact and contact components, and hints are provided to understand the spin-delocalisation mechanism in S=2 systems by keeping in mind the delocalisation patterns in low-spin S=1/2 and high-spin S= 5/2 iron(III) systems.

Anisotropy↗
PubMed2003

A high-resolution NMR study of long-lived water molecules in both oxidation states of a minimal cytochrome c.

The interaction of water with oxidized and reduced cytochrome c from the Gram-positive bacterium Bacillus pasteurii (a 71-amino acid long monoheme cytochrome) is investigated through CLEANEX experiments and (15)N-edited ePHOGSY and Tr-ROESY experiments. It appears that a water molecule gives rise to dipolar cross-relaxation with the amide protons of Gly74 and Ile75, with a residence time longer than 0.4 ns, to account for a negative NOE. Such water molecule is present in both the oxidized and reduced species and in the X-ray structure. It appears to have a structural role. Other possible roles are discussed by comparison with the water molecules present in other c-type cytochromes. The amide proton of Cys35 is found to exchange rapidly with the solvent in the oxidized but not in the reduced protein, at variance with H/D exchange experiments, which probe a different time scale. The present data confirm that electron-transfer proteins evolved to minimize reorganization energy upon change of the oxidation state, even though the consequent variation of charge of the metal ion may induce some changes in the structure and/or dynamics of the protein.

Bacillus↗
PubMed2003

Bioinorganic chemistry in the postgenomic era.

Genome sequencing has revolutionized all fields of life sciences. Bioinorganic chemistry is certainly not immune to this influence, which is presenting unprecedented challenges. A new goal for bioinorganic chemistry is the investigation of the linkages between inorganic elements and genomic information. This requires new advancements andor the development of new expertise in fields such as bioinformatics and genetics but also provides a driving force to push forward the exploitation of traditional analytical techniques and spectroscopic tools. The "case study" of metal homeostasis in cells is discussed to provide a flavor of the current evolution of the field.

Base Sequence↗
PubMed2003

A redox switch in CopC: an intriguing copper trafficking protein that binds copper(I) and copper(II) at different sites.

The protein CopC from Pseudomonas syringae has been found capable of binding copper(I) and copper(II) at two different sites, occupied either one at a time or simultaneously. The protein, consisting of 102 amino acids, is known to bind copper(II) in a position that is now found consistent with a coordination arrangement including His-1, Glu-27, Asp-89, and His-91. A full solution structure analysis is reported here for Cu(I)-CopC. The copper(I) site is constituted by His-48 and three of the four Met residues (40, 43, 46, 51), which are clustered in a Met-rich region. Both copper binding sites have been characterized through extended x-ray absorption fine structure studies. They represent novel coordination environments for copper in proteins. The two sites are approximately 30 A far apart and have little affinity for the ion in the other oxidation state. Oxidation of Cu(I)-CopC or reduction of Cu(II)-CopC causes migration of copper from one site to the other. This behavior is observed both in NMR and EXAFS studies and indicates that CopC can exchange copper between two sites activated by a redox switch. CopC resides in the periplasm of Gram-negative bacteria where there is a multicopper oxidase, CopA, which may modulate the redox state of copper. CopC and CopA are coded in the same operon, responsible for copper resistance. These peculiar and novel properties of CopC are discussed with respect to their relevance for copper homeostasis.

Amino Acid Sequence↗
PubMed2003

Structural model for an alkaline form of ferricytochrome C.

An (15)N-enriched sample of the yeast iso-1-ferricytochrome c triple variant (Lys72Ala/Lys79Ala/Cys102Thr) in an alkaline conformation was examined by NMR spectroscopy. The mutations were planned to produce a cytochrome c with a single conformer. Despite suboptimal conditions for the collection of spectra (i.e., pH approximately equal to 11), NMR remains a suitable investigation technique capable of taking advantage of paramagnetism. 76% of amino acids and 49% of protons were assigned successfully. The assignment was in part achieved through standard methods, in part through the identification of groups maintaining the same conformation as in the native protein at pH 7 and, for a few other residues, through a tentative analysis of internuclear distance predictions. Lys73 was assigned as the axial ligand together with His18. In this manner, 838 meaningful NOEs for 108 amino acids, 50 backbone angle constraints, and 203 pseudocontact shifts permitted the convergence of randomly generated structures to a family of conformers with a backbone RMSD of 1.5 +/- 0.2 A. Most of the native cytochrome c conformation is maintained at high pH. The NOE pattern that involves His18 clearly indicates that the proximal side of the protein, including the 20s and 40s loops, remains essentially intact. Structural differences are concentrated in the 70-80 loop, because of the replacement of Met80 by Lys73 as an axial ligand, and in the 50s helix facing that loop; as a consequence, there is increased exposure of the heme group to solvent. Based on several spectral features, we conclude that the folded polypeptide is highly fluxional.

Cytochromes c↗
PubMed2003

X-ray absorption and NMR spectroscopic studies of CopZ, a copper chaperone in Bacillus subtilis: the coordination properties of the copper ion.

XAS studies have been performed, under various experimental conditions, on a copper(I)-transporting protein, CopZ, of Bacillus subtilis. The copper(I) ion, reduced with dithiothreitol, is three-coordinate with three sulfur donor atoms, two of which presumably provided by the protein and one by dithiothreitol. If a molar excess of acetate (15 mM; 5:1 respect to CopZ) or citrate (6 mM; 2:1 respect to CopZ) is present in solution, the EXAFS spectra suggest the presence of a dimeric form involving a close contact between Cu(I) ions from two molecules, where Cu is still three-coordinate. (1)H and (15)N NMR data provide further structural details. If copper reduction is accomplished with ascorbate, the data indicate that one oxygen of ascorbate enters in the first-coordination sphere of copper, together with two sulfur atoms, in a dimeric form of the protein. These results are instructive and have been discussed with respect to the molecular basis of copper trafficking.

Bacillus subtilis↗
PubMed2003

Dynamic properties of the G93A mutant of copper-zinc superoxide dismutase as detected by NMR spectroscopy: implications for the pathology of familial amyotrophic lateral sclerosis.

The backbone assignment of the copper-zinc superoxide dismutase amyotrophic lateral sclerosis G93A mutant was performed on an (15)N-enriched protein sample. (15)N R(1), R(2), and R(1)(rho) and (15)N-(1)H NOE experiments were then carried out at 600 MHz on G93A Cu(2)Zn(2)SOD and the values compared to the dynamics data for the "wild-type" protein. In addition, (15)N and (1)H chemical shift comparisons between wild-type Cu(2)Zn(2)SOD and its G93A mutant were also made. G93A exhibits a higher mobility than wild-type Cu(2)Zn(2)SOD, particularly in loops III and V, on a time scale faster than the rate of protein tumbling. There are also distinct chemical shift and NOE differences in residues 35-42 and 92-95, which comprise these loops. These two regions of Cu(2)Zn(2)SOD form the end of the beta-barrel termed the "beta-barrel plug" [Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. (1982) J. Mol. Biol. 160, 181-217]. The increased mobility and reduction of the number of observed NOEs in this region indicate an opening of the beta-barrel that may lead to amyloid fibrillogenesis. Alternatively, a motor neuron-specific substrate may bind this region of the protein, leading to deleterious modifications and/or reactions.

Alanine↗
PubMed2003

Understanding copper trafficking in bacteria: interaction between the copper transport protein CopZ and the N-terminal domain of the copper ATPase CopA from Bacillus subtilis.

In this paper the interaction of cytoplasmic CopZ and the N-terminal domain of the CopA ATPase from Bacillus subtilis has been studied by NMR through (15)N-(1)H HSQC experiments in order to understand the role of the two proteins in the whole copper trafficking mechanism of the bacteria. It appears that the two proteins interact in a fashion similar to that of the yeast homologue proteins [Arnesano, F., Banci, L., Bertini, I., Cantini, F., Ciofi-Baffoni, S., Huffman, D. L., and O'Halloran, T. V. (2001) J. Biol. Chem. 276, 41365-41376], although the surface potentials are reversed. A structural model for the interaction is proposed. (15)N mobility studies on the free proteins and on their complex are also reported. From these data, it appears that copper is largely transferred from CopZ to CopA, thus suggesting their possible involvement in a detoxification process. Comparing functional data of homologous proteins of other bacteria, it can be concluded that this class of proteins is involved in copper homeostasis but the specific roles are species dependent.

Adenosine Triphosphatases↗
PubMed2003

Structure and dynamics of reduced Bacillus pasteurii cytochrome c: oxidation state dependent properties and implications for electron transfer processes.

The solution structure of reduced Bacillus pasteurii cytochrome c, which has only 71 amino acids, has been determined by NMR to an RMSD of 0.46 +/- 0.08 A for all backbone atoms and 0.79 +/- 0.08 A for all heavy atoms and refined through restrained energy minimization. The target function out of 1645 constraints is 0.52 +/- 0.11 A(2), and the penalty function is 66 +/- 12 kJ mol(-)(1). The structure appears very similar to that in the oxidized state, only Trp87 and the propionates showing significant differences. The mobility was investigated through (15)N R(1) and R(2) relaxation rates, (15)N-(1)H NOE, and (1)H/(2)H exchange. It is found that the oxidized form is generally more mobile than the reduced one. By comparing the redox-state dependence of the structural/dynamic properties of Fe-S proteins, cytochrome c, and blue copper proteins, hints are provided for a better comprehension of the electron transfer processes.

Amides↗
PubMed2003

Solution structure of Sco1: a thioredoxin-like protein Involved in cytochrome c oxidase assembly.

Sco1, a protein required for the proper assembly of cytochrome c oxidase, has a soluble domain anchored to the cytoplasmic membrane through a single transmembrane segment. The solution structure of the soluble part of apoSco1 from Bacillus subtilis has been solved by NMR and the internal mobility characterized. Its fold places Sco1 in a distinct subgroup of the functionally unrelated thioredoxin proteins. In vitro Sco1 binds copper(I) through a CXXXCP motif and possibly His 135 and copper(II) in two different species, thus suggesting that copper(II) is adventitious more than physiological. The Sco1 structure represents the first structure of this class of proteins, present in a variety of eukaryotic and bacterial organisms, and elucidates a link between copper trafficking proteins and thioredoxins. The availability of the structure has allowed us to model the homologs Sco1 and Sco2 from S. cerevisiae and to discuss the physiological role of the Sco family.

Amino Acid Motifs↗
PubMed2003

Application of NMRD to hydration of rubredoxin and a variant containing a (Cys-S)3FeIII(OH) site.

Hydration of oxidized rubredoxin (Fe(III)(S-Cys)(4) center) was investigated by (1)H and (17)O relaxation measurements of bulk water as a function of the applied magnetic field (nuclear magnetic relaxation dispersion). Oxidized rubredoxin showed an increased water (1)H relaxation profile with respect to the diamagnetic gallium derivative or reduced species. Analysis of the data shows evidence of exchangeable proton(s) approximately 4.0-4.5 A from the metal ion, the exchange time being longer than 10(-10) s and shorter than 10(-5) s. The correlation time for the proton-electrons interaction is 7 x 10(-11) s and is attributed to the effective electron relaxation time. Its magnitude is consistent with the large signal linewidths of the protein donor nuclei, observed in high resolution NMR spectra. For reduced rubredoxin, such correlation time is proposed to be smaller than 10(-11) s. (17)O relaxation measurements suggest the presence of at least one long-lived protein-bound water molecule. Analogous relaxation measurements were performed on the C6S rubredoxin variant, whose iron(III) center has been previously shown to be coordinated to three cysteine residues and a hydroxide ion above pH 6. (1)H nuclear magnetic relaxation dispersion profiles indicate increased hydration with respect to the wild-type.

Binding Sites↗
PubMed2003

15N-1H Residual dipolar coupling analysis of native and alkaline-K79A Saccharomyces cerevisiae cytochrome c.

Residual dipolar couplings (RDCs) and pseudocontact shifts are experimentally accessible properties in nuclear magnetic resonance that are related to structural parameters and to the magnetic susceptibility anisotropy. We have determined RDCs due to field-induced orientation of oxidized-K79A and reduced cytochrome c at pH 7.0 and oxidized-K79A cytochrome c at pH 11.1 through measurements of amide (15)N-(1)H (1)J couplings at 800 and 500 MHz. The pH 7.0 RDCs for Fe(III)- and Fe(II)-cytochrome c together with available nuclear Overhauser effects were used to recalculate solution structures that were consistent with both sets of constraints. Molecular magnetic susceptibility anisotropy values were calculated for both redox states of the protein. By subtracting the residual dipolar couplings (RDCs) of the reduced form from those of the oxidized form measured at the same magnetic field (800 MHz), we found the RDC contribution of the paramagnetic metal ion in the oxidized protein. The magnetic susceptibility anisotropy, which was calculated from the structure, was found to be the same as that of the paramagnetic metal ion obtained independently from pseudocontact shifts, thereby indicating that the elements of secondary structure either are rigid or display the same mobility in both oxidation states. The residual dipolar coupling values of the alkaline-K79A form are small with respect to those of oxidized native cytochrome, whereas the pseudocontact shifts are essentially of the same magnitude, indicating local mobility. Importantly, this is the first time that mobility has been found through comparison of RDCs with pseudocontact shifts.

Anisotropy↗
PubMed2003

A use of Ramachandran potentials in protein solution structure determinations.

A strategy is developed to use database-derived phi-psi constraints during simulated annealing procedures for protein solution structure determination in order to improve the Ramachandran plot statistics, while maintaining the agreement with the experimental constraints as the sole criterion for the selection of the family. The procedure, fully automated, consists of two consecutive simulated annealing runs. In the first run, the database-derived phi-psi constraints are enforced for all amino acids (but prolines and glycines). A family of structures is then selected on the ground of the lowest violations of the experimental constraints only, and the phi-psi values for each residue are examined. In the second and final run, the database-derived phi-psi constraints are enforced only for those residues which in the first run have ended in one and the same favored phi-psi region. For residues which are either spread over different favored regions or concentrated in disallowed regions, the constraints are not enforced. The final family is then selected, after the second run, again only based on the agreement with the experimental constraints. This automated approach was implemented in DYANA and was tested on as many as 12 proteins, including some containing paramagnetic metals, whose structures had been previously solved in our laboratory. The quality of the structures, and of Ramachandran plot statistics in particular, was notably improved while preserving the agreement with the experimental constraints.

Computer Simulation↗
PubMed2002

Solution structure and characterization of the heme chaperone CcmE.

The covalent attachment of the heme cofactor in c-type cytochromes is a surprisingly complex process, which in bacteria involves a number of different proteins. Among the latter, the ccmE gene product is known to perform a key role in the heme delivery pathway in Gram-negative bacteria. The solution structure of the soluble domain of apo-CcmE from Shewanella putrefaciens was determined through NMR spectroscopy on a 13C,15N-labeled sample. The structure is characterized by a compact core with large regions of beta structure, while the N-terminal and C-terminal regions are essentially unstructured. The overall folding is similar to that of the so-called oligo-binding proteins (OB fold). Solvent-exposed aromatic residues, conserved in all CcmE homologues, have been found in the proximity of His131, the putative heme-binding residue, that could have a role in the interaction with heme. No interaction between CcmE and heme, as well as between CcmE and holocytochrome c, could be detected in vitro by electronic spectroscopy or by NMR. The data available suggest that the heme transfer process is likely to involve a heterooligomeric protein complex and occur under a tight enzymatic control.

Aspartate-tRNA Ligase↗
PubMed2002

A new zinc-protein coordination site in intracellular metal trafficking: solution structure of the Apo and Zn(II) forms of ZntA(46-118).

Zinc, a metal ion that functions in a wide variety of catalytic and structural sites in metalloproteins, is shown here to adopt a novel coordination environment in the Escherichia coli transport protein ZntA. The ZntA protein is a P-type ATPase that pumps zinc out of the cytoplasm and into the periplasm. It is physiologically selective for Zn(II) and functions with metalloregulatory proteins in the cell to keep the zinc quota within strict limits. Yet, the N-terminal cytoplasmic domain contains a region that is highly homologous to the yeast Cu(I) metallochaperone Atx1. To investigate how the structure of this region may influence its function, this fragment, containing residues 46-118, has been cloned out of the gene and overexpressed. We report here the solution structure of this fragment as determined by NMR. Both the apo and Zn(II)-ZntA(46-118) structures have been determined. It contains a previously unknown protein coordination site for zinc that includes two cysteine residues, Cys59 and Cys62, and a carboxylate residue, Asp58. The solvent accessibility of this site is also remarkably high, a feature that increasingly appears to be a characteristic of domains of heavy metal ion transport proteins. The participation of Asp58 in this ZntA metal ion binding site may play an important role in modulating the relative affinities and metal exchange rates for Zn(II)/Pb(II)/Cd(II) as compared with other P-type ATPases, which are selective for Cu(I) or Ag(I).

Adenosine Triphosphatases↗
PubMed2002

The unfolding of oxidized c-type cytochromes: the instructive case of Bacillus pasteurii.

The reversible unfolding of oxidized Bacillus pasteurii cytochrome c(553) by guanidinium chloride under equilibrium conditions has been monitored by NMR and optical spectroscopy. The results obtained indicate that unfolding takes place through a mechanism involving the detachment from heme iron coordination of the sulfur of the Met71 axial ligand and yielding either a high spin (HS) or a low spin (LS(1)) species, depending on the pH value. In the LS(1) form the Met71 is replaced by another protein ligand, possibly Lys. The ligand exchange reaction does not reach completion until the protein backbone reaches a largely unfolded state, as monitored through 1H-15N NMR experiments, thus demonstrating that there is a significant correlation between formation of the Fe-S bond and native structure stability. 1H/2H exchange data, however, show that helix alpha(3), the C-terminal region of helix alpha(4), and helix alpha(5) maintain low exchangeability of the amide protons in the LS(1) form. This finding most likely implies that these regions maintain some ordered non-covalent structure, in which the amide moieties are involved in H-bonds. Finally, a folding mechanism is proposed and discussed in terms of analogies and differences with the larger mitochondrial cytochrome c proteins. It is concluded that the thermodynamic stability of the region around the metal cofactor is determined by the chemical nature of the residues around the axial methionine residue.

Bacillus↗
PubMed2002

The metal reductase activity of some multiheme cytochromes c: NMR structural characterization of the reduction of chromium(VI) to chromium(III) by cytochrome c(7).

The redox reaction between CrO(4)(2-) and the fully reduced three-heme cytochrome c(7) from Desulfuromonas acetoxidans to give chromium(III) and the fully oxidized protein has been followed by NMR spectroscopy. The hyperfine coupling between the oxidized protein protons and chromium(III), which remains bound to the protein, gives rise to line-broadening effects on the NMR resonances that can be transformed into proton-metal distance restraints. Structure calculations based on these unconventional constraints allowed us to demonstrate that chromium(III) binds at a unique site and to locate it on the protein surface. The metal ion is located 7.9 +/- 0.4 A from the iron of heme IV, 16.3 +/- 0.7 A from the iron of heme III, and 22.5 +/- 0.5 A from the iron of heme I. Shift changes caused by the presence of unreactive MoO(4)(2-), a CrO(4)(2-) analogue, indicate the involvement of the same protein area in the anion binding. The titration of the oxidation of cytochrome c(7) shows a detailed mechanism of action. The presence of a specific binding site supports the hypothesis of the biological role of this cytochrome as a metal reductase.

Binding Sites↗