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Ivano Bertini

Publications and source records attributed to Ivano Bertini.

At least 19 recordsLinked to original sources
PubMed2004

Cytochrome c and SDS: a molten globule protein with altered axial ligation.

Saccharomices cerevisiae (yeast iso-1) cytochrome c has been investigated in the presence of 100 mM SDS in order to simulate the interaction of cytochrome c with membrane. Under these circumstances, a high spin species with detached methionine axial ligand is observed through NMR, in analogy to findings on the horse heart protein. However, at variance with the latter system, for the yeast protein also a low spin species is detected, which appears to be present with a concentration of about 40% with respect to that of the high spin species. The R(1), R(2), [1H]-15N NOE of backbone amides which are not affected by paramagnetism are homogeneous and allow a simultaneous analysis of the data for the two species. The result is that the rotational correlation time is larger than in water and larger than expected on the basis of viscosity of the SDS-containing solution. This finding suggests interactions of cytochrome c with SDS. Furthermore, it appears that there is subnanosecond backbone mobility, which also accounts for the decreased intensity of NOE cross-peaks and may be associated with equilibria between helical and random coil structure. The dynamic behavior appears to be a common feature of the high spin and low spin species and is consistent with the presence of a molten globule state. The molten globule nature of the protein could account for the presence of the different axial coordination of the heme iron. Such findings are meaningful with respect to the physiology of cytochrome c as electron transfer protein and as promoter of apoptosis.

Animals↗
PubMed2004

13C-13C NOESY: an attractive alternative for studying large macromolecules.

13C direct detection provides a valuable alternative to 1H detection to overcome fast relaxation because of its smaller magnetic moment. 13C-13C NOESY spectra were acquired for a dimeric protein of molecular mass 32 000 and for a monomeric analogue. With increasing molecular mass, the quality of 13C-13C NOESY spectra improves while the scalar-based experiments become less sensitive, as predicted by the increase in the molecular mass. 13C-13C NOESY spectra of the dimer were acquired with different mixing times. The mixing time can be tuned to detect mainly one-bond correlations, or it can be increased to also detect correlations between nuclei at longer distances. It is proposed that 13C-13C dipolar-based experiments provide a promising tool for signal detection and assignment in large macromolecules, such as multimeric species and macromolecular complexes, for which scalar-based experiments become less effective.

Carbon Isotopes↗
PubMed2004

NMR-validated structural model for oxidized Rhodopseudomonas palustris cytochrome c(556).

The structure of oxidized Rhodopseudomonas palustris cytochrome c(556) has been modeled after that of high-spin cytochrome c' from the same bacterium, the latter being the protein with the greatest sequence identity (35%) among all sequenced proteins in the genomes. The two proteins differ in the number of ligands to iron and in spin state, the former being six-coordinate low-spin and the latter five-coordinate high-spin. In order to validate this modeled structure, several structural restraints were obtained by performing a restricted set of NMR experiments, without performing a complete assignment of the protein signals. The aim was to exploit the special restraints arising from the paramagnetism of the metal ion. A total of 43 residual-dipolar-coupling and 74 pseudocontact-shift restraints, which together sampled all regions of the protein, were used in conjunction with over 40 routinely obtained NOE distance restraints. A calculation procedure was undertaken combining the program MODELLER and the solution structure determination program PARAMAGNETIC DYANA, which includes paramagnetism-based restraints. The directions and magnitude of the magnetic susceptibility anisotropy tensor were also calculated. The approach readily provides useful results, especially for paramagnetic metalloproteins of moderate to large dimensions.

Algorithms↗
PubMed2004

Paramagnetism-based restraints for Xplor-NIH.

Modules that use paramagnetism-based NMR restraints have been developed and integrated in the well known program for solution structure determination Xplor-NIH; the complete set of such modules is called PARArestraints for Xplor-NIH. Paramagnetism-based restraints are paramagnetic relaxation enhancements, pseudocontact shifts, residual dipolar couplings due to metal and overall magnetic anisotropy, and cross correlation between Curie relaxation and nuclear-nuclear dipolar relaxation. The complete program has been tested by back-calculating NOEs and paramagnetism-based restraints from the X-ray structure of cytochrome c (553) from B. pasteurii. Furthermore, the same experimental restraints previously used to determine the solution structure of cytochrome c (553) itself, of cytochrome b (5), and of calbindin D(9k) with the program PARAMAGNETIC DYANA, have been used for structure calculations by using PARArestraints for Xplor-NIH. The agreement between the two programs is quite satisfactory and validates both protocols.

Calbindins↗
PubMed2003

13C direct detection experiments on the paramagnetic oxidized monomeric copper, zinc superoxide dismutase.

In this report, the use of 13C direct detection has been pursued in 2D experiments (13C-13C COSY, 13C-13C COCAMQ, 13C-13C NOESY) to detect broad lines in nuclear magnetic resonance spectra of paramagnetic metalloproteins. The sample is a monomeric oxidized copper, zinc superoxide dismutase. Thanks to direct detection probeheads, cryogenic technology, and implementation of 13C band-selective homodecoupling, many broadened signals were detected. Proton signals for the same residues escaped detection in 1H and 1H-15N HSQC experiments because of the broadening. Only the 13C signals which experience large contact coupling escaped detection, i.e., the 13C nuclei of the metal coordinated histidines. Otherwise, nuclei as close to copper(II) as 4 A can be detected. Paramagnetic-based restraints can in principle be used for solution structure determination of paramagnetic metalloproteins and in copper(II) proteins in particular. The present study is significant also for the study of large diamagnetic proteins for which proton relaxation makes proton-based spectroscopy not adequate.

Carbon Isotopes↗
PubMed2003

Solution structure of apo CopZ from Bacillus subtilis: further analysis of the changes associated with the presence of copper.

The solution structure of apo CopZ from Bacillus subtilis has been determined with the aim of investigating the changes in the hydrophobic interactions around the M-X-C-X-X-C copper(I) binding motif upon metal binding. The methionine of this motif (Met 11 in CopZ) points toward the solvent in apo CopZ, whereas its sulfur atom is close to the metal ion in the metal-loaded protein, though probably not at binding distance. This change is associated with the weakening of the interaction between Leu 37 and Cys 16, present in the apo form, and the formation of an interaction between Met 11 and Tyr 65. Loops 1, 3, and 5 are affected by metal binding. Comparison with the structure of other homologous proteins confirms that often metal binding affects a hydrophobic patch around the metal site, possibly for optimizing and tuning the hydrophobic interactions with the partners. It is also shown that copper(I) exchanges among apo CopZ molecules in slow exchange on the NMR time scale, whereas it is known that such exchange between partner molecules (i.e., metallochaperones and metal pumps) is fast.

Amino Acid Motifs↗
PubMed2003

Structural basis for the function of the N-terminal domain of the ATPase CopA from Bacillus subtilis.

The solution structure of the N-terminal region (151 amino acids) of a copper ATPase, CopA, from Bacillus subtilis, is reported here. It consists of two domains, CopAa and CopAb, linked by two amino acids. It is found that the two domains, which had already been separately characterized, interact one to the other through a hydrogen bond network and a few hydrophobic interactions, forming a single rigid body. The two metal binding sites are far from one another, and the short link between the domains prevents them from interacting. This and the surface electrostatic potential suggest that each domain receives copper from the copper chaperone, CopZ, independently and transfers it to the membrane binding site of CopA. The affinity constants of silver(I) and copper(I) are similar for the two sites as monitored by NMR. Because the present construct "domain-short link-domain" is shared also by the last two domains of the eukaryotic copper ATPases and several residues at the interface between the two domains are conserved, the conclusions of the present study have general validity for the understanding of the function of copper ATPases.

Adenosine Triphosphatases↗
PubMed2003

The evolutionarily conserved trimeric structure of CutA1 proteins suggests a role in signal transduction.

CutA1 are a protein family present in bacteria, plants, and animals, including humans. Escherichia coli CutA1 is involved in copper tolerance, whereas mammalian proteins are implicated in the anchoring of acetylcholinesterase in neuronal cell membranes. The x-ray structures of CutA1 from E. coli and rat were determined. Both proteins are trimeric in the crystals and in solution through an inter-subunit beta-sheet formation. Each subunit consists of a ferredoxin-like (beta1alpha1beta2beta3alpha2beta4) fold with an additional strand (beta5), a C-terminal helix (alpha3), and an unusual extended beta-hairpin involving strands beta2 and beta3. The bacterial CutA1 is able to bind copper(II) in vitro through His2Cys coordination in a type II water-accessible site, whereas the rat protein precipitates in the presence of copper(II). The evolutionarily conserved trimeric assembly of CutA1 is reminiscent of the architecture of PII signal transduction proteins. This similarity suggests an intriguing role of CutA1 proteins in signal transduction through allosteric communications between subunits.

Allosteric Site↗
PubMed2003

Thermotoga maritima IscU. Structural characterization and dynamics of a new class of metallochaperone.

Members of the IscU family of proteins are among the most conserved of all protein groups, extending across all three kingdoms of life. IscU serves as a scaffold for the assembly of intermediate iron-sulfur cluster centers and further mediates delivery to apo protein targets. Several proteins that mediate delivery of single metal ions to apo targets (termed metallochaperones) have recently been characterized structurally. Each displays a ferredoxin-like betaalphabetabetaalphabeta motif as a structural core. Assembly and delivery of a polynuclear iron-sulfur cluster is, however, a more complex pathway and presumably would demand a distinctive protein mediator. Here, we demonstrate Thermotoga maritima IscU (Tm IscU) to display unique structural and motional characteristics that distinguish it from other members of this class of proteins. In particular, IscU adopts a mobile, physiologically relevant, molten globule-like state that is vastly different from the previously identified ferredoxin-like fold that has thus far been characterized for other metallochaperones. The secondary structural content of Tm IscU is consistent with previous circular dichroism measurements on apo and holo protein, consisting of six alpha-helices and three beta-strands, the latter forming an anti-parallel beta-sheet. Extensive dynamics studies are consistent with a protein that has reasonably well defined secondary structural elements, but with a tertiary structure that is fluxional among widely different conformational arrangements. Analogous conformational flexibility does not exist in other structurally characterized metallochaperones; however, such a dynamic molecule may account for the lack of long-range NOEs, and allow both for the flexibility that is necessary for the multiple roles of Fe-S cluster assembly, and recognition and delivery of that cluster to a target protein. Additionally, the fluxionality of IscU is unique in that the protein appears to be more compact (based on 1H/2H exchange, R1, R2, and NOE data) but yet more fluid (lack of long-range NOEs) than typical molten globule proteins.

Amino Acid Motifs↗
PubMed2003

Solution structure of Apo Cu,Zn superoxide dismutase: role of metal ions in protein folding.

The solution structure of the demetalated copper, zinc superoxide dismutase is obtained for the monomeric Glu133Gln/Phe50Glu/Gly51Glu mutant through NMR spectroscopy. The demetalated protein still has a well-defined tertiary structure; however, two beta-strands containing two copper ligands (His46 and His48, beta4) and one zinc ligand (Asp83, beta5) are shortened, and the sheet formed by these strands and strands beta7 and beta8 moves away from the other strands of the beta-barrel to form an open clam with respect to a closed conformation in the holoprotein. Furthermore, loop IV which contains three zinc ligands (His63, His71, and His80) and loop VII which contributes to the definition of the active cavity channel are severely disordered, and experience extensive mobility as it results from thorough (15)N relaxation measurements. These structural and mobility data, if compared with those of the copper-depleted protein and holoprotein, point out the role of each metal ion in the protein folding, leading to the final tertiary structure of the holoprotein, and provide hints for the mechanisms of metal delivery by metal chaperones.

Amino Acid Substitution↗
PubMed2003

A core mutation affecting the folding properties of a soluble domain of the ATPase protein CopA from Bacillus subtilis.

The two N-terminal domains of the P-type copper ATPase, CopAa and CopAb, from Bacillus subtilis differ in their folding capabilities in vitro. Whereas CopAb has the typical betaalphabetabetaalphabeta structure and is a rigid protein, CopAa is found to be largely unfolded. A sequence analysis of the two and of orthologue homologous proteins indicates that Ser46 in CopAa may destabilise the hydrophobic core, as also confirmed through a bioinformatic energy study. CopAb has a Val in the corresponding position. The S46V and S46A mutants are found to be folded, although the latter displays multiple conformations. S46VCopAa, in both apo and copper(I) loaded forms, has very similar structural and dynamic properties with respect to CopAb, besides a different length of strand beta2 and beta4. It is intriguing that the oxygen of Thr16 is found close, though at longer than bonding distance, to copper in both domains, as it also occurs in a human orthologue domain. This study contributes to understanding the behaviour of proteins that do not properly fold in vitro. A possible biological significance of the peculiar folding behaviour of this domain is discussed.

Adenosine Triphosphatases↗
PubMed2003

A further investigation of the cytochrome b5-cytochrome c complex.

The interaction of reduced rabbit cytochrome b(5) with reduced yeast iso-1 cytochrome c has been studied through the analysis of (1)H-(15)N HSQC spectra, of (15)N longitudinal ( R(1)) and transverse ( R(2)) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b(5) has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b(5).

Animals↗
PubMed2003

Tuning the affinity for lanthanides of calcium binding proteins.

The possibility of selectively substituting one or more lanthanides into the four canonical calcium binding sites of calcium-loaded vertebrate calmodulin (CaM) was investigated by monitoring changes in the (1)H-(15)N HSQC NMR spectra of the (15)N-enriched protein upon titration with Yb(3+). The affinity of lanthanides for both N-terminal sites I and II is only moderately higher than that of calcium, and comparable with that of calcium for the two C-terminal sites. This situation induces binding of lanthanides to other noncanonical sites located at the interdomain linker, the N- and C-terminal ends, and at the inter-EF-hand linkers. Therefore, mutants were designed to alter the metal binding properties of calcium sites I (D22N, D24E), II (D58N, N60D, D58N-N60D), III (N97D), II-III (N60D-N97D), and IV (D129N), as well as of the inter-EF-hand linker of the N-terminal domain (N42K, T44K). The most striking effects were obtained for the N60D mutant at site II, as selective lanthanide binding is achieved even in the presence of excess calcium, and little or no population of the noncanonical sites occurs. Similar although less pronounced effects were observed for the N97D mutant. These findings allow us to better define some of the determinants of the relative affinities of calcium and lanthanides in CaM and, by extension, in other calcium binding proteins. Finally, by using CaM samples containing only three of the four calcium ions, it was possible to prepare well-defined Ca(3)Ln-CaM derivatives (Ln = Tb, Dy, Tm, and Yb), with interesting properties as NMR probes.

Animals↗
PubMed2003

Superoxide dismutase folding/unfolding pathway: role of the metal ions in modulating structural and dynamical features.

The unfolding and refolding of a monomeric mutant of copper-zinc superoxide dismutase was investigated by NMR spectroscopy in the copper-reduced form and by using guanidinium chloride as denaturing agent. It is found that the protein gives rise to a series of intermediates at low guanidinium concentration and to a globular unfolded state at a guanidinium concentration higher than 3.5 M, which closely resembles a random coil structure, but with a high degree of compactness. At 1.9 M guanidinium, the intermediate and unfolded forms are present in equimolar ratio. The intermediate states show changes in the 1H and 15N chemical shifts with respect to the native protein. The perturbations on the signals occur at different GdmCl concentrations for different regions of the protein. The residues affected first are located in the loops and in the beta3 strand, followed by changes in the sheet formed by beta4, beta5, beta7, beta8 strands. The transition into the unfolded structure implies the detachment of the metal ions from the native coordination sites, even if non-specific interactions with the metal ions remain. R(1), R(2), [1H]-15N NOE, and CLEANEX experiments provide information on the mobility at the various stages showing how protein rigidity is lost during unfolding. The whole process is reversible. The oxidized species behaves in a similar way. The apo protein shows formation of 50% of the unfolded species at a guanidinium concentration of 0.4 M, thus demonstrating the importance of metal ions with respect to the unfolding process and protein structure stability. Hints to understand the whole folding process are obtained and discussed.

Circular Dichroism↗
PubMed2003

A strategy for the NMR characterization of type II copper(II) proteins: the case of the copper trafficking protein CopC from Pseudomonas Syringae.

CopC from Pseudomonas syringae was found to be a protein capable of binding both Cu(I) and Cu(II) at two different sites. The solution structure of the apo protein is available, and structural information has been obtained on the Cu(I) bound form. We attempt here to set the limits for the determination of the solution structure of a Cu(II) protein, such as the Cu(II) bound form of CopC, in which the Cu(II) ion takes a type II coordination. The electron relaxation time is estimated from NMRD measurements to be 3 ns which leads to a correlation time for the nuclear spin-electron spin dipolar interaction of 2 ns. This information allowed us to tailor the NMR experiments and to fully exploit purely heteronuclear spectroscopy to assign as many signals as possible. In this way, 37 (13)C and 11 (15)N signals that completely escape detection with conventional approaches were assigned. Paramagnetic based structural constraints were obtained by measuring paramagnetic longitudinal relaxation enhancements (rho(para)) which allowed us to precisely locate the copper ion within the protein frame. Pseudocontact shifts (pcs's) were also used as constraints for 83 (1)H and 18 (13)C nuclei. With them, together with other standard structural constraints, a structure is obtained (and submitted to PDB) where information is only missing in a sphere with a 6 A radius from the copper ion. If we borrow information from EXAFS data, which show evidence of two copper coordinated histidines, then His 1 and His 91 are unambiguously identified as copper ligands. EXAFS data indicate two more light donor atoms (O/N) which could be from Asp 27 and Glu 89, whereas the NMRD data indicate the presence of a semicoordinated water molecule at 2.8 A (Cu-O distance) roughly orthogonal to the plane identified by the other four ligands. This represents the most extensively characterized structure of a type II Cu(II) protein obtained employing the most advanced NMR methods and with the aid of EXAFS data. The knowledge of the location of the Cu(II) in the protein is important for the copper transfer mechanism.

Bacterial Proteins↗
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↗