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I Bertini

Publications and source records attributed to I Bertini.

At least 109 records · Page 6Linked to original sources
PubMed1996

The solution structure refinement of the paramagnetic reduced high-potential iron-sulfur protein I from Ectothiorhodospira halophila by using stable isotope labeling and nuclear relaxation.

The reduced high-potential iron sulfur protein I from Ectothiorhodospira halophila which contains the [4Fe-4S]2+ polymetallic center has been fully labeled with 15N and 13C. The protein is paramagnetic, the nuclear relaxation times of nuclei close to the paramagnetic ion are drastically shortened and some strategic dipolar connectivities are lost. Notwithstanding, the solution structure has been reported [Banci, L., Bertini, I., Eltis, L. D., Felli, I. C., Kastrau, D. H. W., Luchinat, C., Piccioli, M., Pierattelli, R. & Smith, M. (1994) Eur. J. Biochem. 225, 715-725]. We have performed classical HNHA, HNCA soft-COSY, soft-HCCH E. COSY and 15N-1H correlated NOESY experiments in order to obtain a set of 3J scalar coupling constants. Some experiments have been optimized to counterbalance the effect of paramagnetism. From heteronuclear single-quantum experiments preceded by a 180 degrees pulse and variable delay times, the non-selective magnetization recovery has been followed from which the contribution to dipolar relaxation of nuclei due to the interaction with the paramagnetic metal ions (rho para) has been estimated. Finally, the intensities of NOEs have been corrected for the presence of paramagnetic metal ions and these corrected values together with 3J values and rho para data have been used to obtain a well defined solution structure. The aim is that of obtaining a structure with enough constraints to be well resolved all over the protein, including the vicinity of the paramagnetic metal cluster, which is anchored to the protein through the rho para constraints. In total, 1226 corrected NOESY crosspeaks (of which 945 were found to be meaningful), 37 one-dimensional NOEs, 39 3JHNH alpha and 37 3JHNC' (providing 45 phi dihedral angle constraints) 54 3JH alpha H beta and 31 3JNH beta (providing 26 chi 1 dihedral angle constraints), 4 chi 2 dihedral angle constraints of the coordinated cysteines, obtained from the hyperfine shifts of the beta CH protons, and 58 rho para constraints, have been used for structure calculation. Restrained molecular dynamics simulations have also been performed to provide the final family of structures. This research demonstrates that stable isotope labeling provides specific advantages for the NMR investigation of paramagnetic molecules, as the small magnetic moment of heteronuclei minimizes the paramagnetic influence of unpaired electrons.

Amino Acid Sequence↗
PubMed1996

Three-dimensional structure of the reduced C77S mutant of the Chromatium vinosum high-potential iron-sulfur protein through nuclear magnetic resonance: comparison with the solution structure of the wild-type protein.

The full 1H NMR assignment of the reduced C77S mutant of Chromatium vinosum high-potential iron-sulfur protein (HiPIP) was achieved by taking advantage of the assignment available for the wild-type protein. A total of 1565 nuclear Overhauser effect (NOE) spectroscopy cross peaks were integrated and converted into distance constraints, of which 497 were found to be irrelevant. An additional 24 dipolar constraints were obtained from one-dimensional NOE difference spectra by saturating hyperfine-shifted beta CH2 cysteine/serine protons. Forty-six 3JNH-H alpha coupling constants and eight hydrogen bonds provided further constraints. Through a distance geometry approach, a family of 15 structures was calculated, which was subsequently subjected to restrained energy minimization. The root mean square deviations of the minimized structures were 0.62 +/- 0.09 and 1.09 +/- 0.11 A for backbone and heavy atoms, respectively. The resulting solution structures are very similar to those of the reduced wild-type protein (WT). An analysis of the NOEs experienced by the protons of Ser-77 in both the reduced and oxidized forms reveals that they are very similar to those experienced by Cys-77 in WT. On the basis of the hyperfine shifts observed for the Ser-77 protons and of the present structural analysis, it is concluded that the serine O gamma atom is coordinated to the polymetallic center, thus confirming the strict analogy of the electronic structures of the polymetallic center in both proteins. Capillary electrophoresis experiments demonstrate coordination of Ser-77 as an anion. Serine versus cysteine coordination in iron-sulfur proteins is briefly discussed.

Amino Acid Sequence↗
PubMed1996

Paramagnetic NMR analysis of the seven-iron ferredoxin from the hyperthermoacidophilic archaeon Desulfurolobus ambivalens reveals structural similarity to other dicluster ferredoxins.

The seven-iron ferredoxin from the hyperthermophilic archaeon Desulfurolobus ambivalens has been investigated by one-dimensional and two-dimensional 1H-NMR in its oxidized and dithionite-reduced states. All iron atoms of both the three-iron and the four-iron cluster are bound to cysteine residues whose hyperfine-shifted resonances were characterized. The pattern of these resonances is similar to those from three-iron, four-iron and eight-iron ferredoxins previously described in the literature, but the four-iron cluster has a shift pattern different from that in other seven-iron proteins. A second set of hyperfine-shifted resonances clearly indicates sample heterogeneity, which possibly involves the four-iron cluster. The observation of interresidue NOEs between two different cysteine residues proves the existence of close spatial proximity of the two clusters in D. ambivalens ferredoxin and therefore indicates structural homology to other dicluster ferredoxins. Moreover, this feature is crucial for the sequence-specific assignment of the hyperfine-shifted resonances. The C alpha-C beta-S-Fe dihedral angles of the cysteine residues coordinating the four-iron cluster could be estimated, and the electronic structure of the three-iron cluster is discussed.

Ferredoxins↗
PubMed1996

A complete relaxation matrix refinement of the solution structure of a paramagnetic metalloprotein: reduced HiPIP I from Ectothiorhodospira halophila.

We have accounted for the effect of paramagnetism on the intensities of NOEs in a 73-residue paramagnetic metalloprotein, the reduced high-potential iron sulfur protein ISO I from Ectothiorhodospira halophila, whose solution structure had been recently solved by us. The paramagnetic effects were dealt with through a suitably modified complete relaxation matrix approach. We have then recalculated the structure through a distance geometry program by minimizing the difference between the sixth roots of the calculated and experimental NOEs. The average RMSD, calculated on residues 4-71, within the structures constituting the two families decreased from 0.67 to 0.46 angstrom for backbone atoms and from 1.23 to 1.06 angstroms for all heavy atoms. The structures in the new family are for the most part within the indetermination of the previous, less resolved, family. A few specific differences are detected and related to the presence of non-negligible paramagnetic effects, which are now properly evaluated.

Bacteria↗
PubMed1995

Synthesis and characterization of a monomeric mutant Cu/Zn superoxide dismutase with partially reconstituted enzymic activity.

A monomeric analog of human Cu/Zn superoxide dismutase (F50E/G51E SOD), previously characterized and found to have reduced enzymic activity, was here further modified by replacing Glu133 with Gln. This substitution does not dramatically affect the coordination geometry at the active site, but enhances enzymic activity, and also increases the affinity for anions at the active site. This behavior parallels earlier published results in which this point mutation was made in the dimeric wild-type enzyme. The analog described here has afforded for the first time a monomeric superoxide dismutase with substantial activity. This point mutation does not significantly influence the protein structure but interactions with anions, including superoxide, are altered with respect to the monomeric form. The present monomeric Glu133Gln mutant has partially restored enzymic activity. The diminished activity of the monomeric analogs is discussed in the light of possible minor structural changes and some of their characteristics are compared with those of naturally occurring mutants associated with various neurological diseases.

Binding Sites↗
PubMed1995

Evidence of histidine coordination to the catalytic ferrous ion in the ring-cleaving 2,2',3-trihydroxybiphenyl dioxygenase from the dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1.

The 1H NMR spectra of an aromatic ring-cleaving extradiol dioxygenase, 2,2',3-trihydroxybiphenyl dioxygenase of the dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1, are reported. In the catalytically active reduced form of the monomeric enzyme (MW = 32 kDa), three broad strongly downfield shifted signals were observed, two of which disappeared in D2O solution. Their shifts and linewidths are consistent with ring NH and meta-like protons of coordinated histidines. These signals show strong sensitivity to the presence of the substrate. The oxidized form of the enzyme shows no hyperfine shifted signals. It is suggested that the high spin Fe(II) ion present in the active form of the enzyme is coordinated by at least two histidines. This is the first report of hyperfine shifted NMR signals being detected for an extradiol dioxygenase.

Benzofurans↗
PubMed1995

pH, electrolyte, and substrate-linked variation in active site structure of the Trp51Ala variant of cytochrome c peroxidase.

Electronic absorption, MCD, and 1H NMR spectroscopy have been used to characterize the structures and linkage relationships of three active site states, LS1, HS, and LS2, of the Trp51Ala variant of yeast cytochrome c peroxidase (CcP) in the Fe(III) state. In addition, the binding of three substrates (styrene, catechol, and guaiacol) to the Fe(III) variant has been studied by 1H NMR spectroscopy, and the paramagnetically shifted resonances of the cyanide adduct of the variant have been assigned. The heme iron is hexacoordinated in all three pH-dependent states of the enzyme. LS1, the dominant acidic species, exhibits electronic and MCD spectra indicative of low-spin, bis-histidine coordination environment for the heme iron. The HS form, which dominates at intermediate pH, exhibits electronic, MCD, and 1H NMR spectra characteristic of high-spin heme Fe(III) with axial histidyl and water ligands. The LS2 species exhibits spectroscopic properties indicative of a bis-histidine, low-spin Fe(III) derivative. The equilibrium constants for interconversion of these forms of the variant enzyme are highly dependent on ionic strength, specific anions, and temperature of the solution, with the HS form stabilized relative to the other forms in the presence of several noncoordinating, anionic species. Aromatic substrates such as styrene, catechol, and guaiacol affect the chemical shifts of the heme substituents of the HS species but not of the LS2 species. Based on these results, a model is proposed that accounts to a large extent for the electrostatic origin of the three forms of the active site of the Trp51Ala variant and the mechanisms by which they are differentially stabilized in solution.

Anions↗
PubMed1995

Three-dimensional solution structure of the cyanide adduct of a Met80Ala variant of Saccharomyces cerevisiae iso-1-cytochrome c. Identification of ligand-residue interactions in the distal heme cavity.

The 1H NMR spectrum of the the cyanide adduct of a triply mutated Saccharomyces cerevisiae iso-1-cytochrome c (His39Gln/Met80Ala/Cys102Ser) in the oxidized form has been assigned through 1D NOE and 2D COSY, TOCSY, NOESY, and NOE-NOESY experiments; 562 protons out of a total of 683 have been assigned. The solution structure, the first of a paramagnetic heme protein, was determined using 1426 meaningful NOE constraints out of a total of 1842 measured NOEs. The RMSD values at the stage of restrained energy minimization of 17 structures obtained from distance geometry calculations are 0.68 +/- 0.11 and 1.32 +/- 0.14 A for the backbone and all heavy atoms, respectively. The quality, in terms of RMSD, of the present structure is the same as that obtained for the solution structure of the diamagnetic horse heart ferrocytochrome c [Qi, P. X., et al. (1994) Biochemistry 33, 6408-6419]. The secondary structure elements and the overall folding in the variant are observed to be the same as those of the wild-type protein for which the X-ray structure is available. However, the replacement of the methionine axial ligand with an alanine residue creates a ligand-binding "distal cavity". The properties of the distal cavity seen in this solution structure are compared to those of other heme proteins.

Alanine↗
PubMed1995

Solution structure of the oxidized 2[4Fe-4S] ferredoxin from Clostridium pasteurianum.

Following the recently developed approach to the solution structure of paramagnetic high-potential iron-sulfur proteins, the three-dimensional structure in solution of the oxidized Clostridium pasteurianum ferredoxin has been solved by 1H-NMR. The X-ray structure is not available. The protein contains 55 amino acids and two [4Fe-4S] clusters. In the oxidized state, the clusters have S = 0 ground states, but are paramagnetic because of thermal population of excited states. Due to the somewhat small size of the protein and to the presence of two clusters, approximately 55% of the residues have at least one proton with a non-selective T1 smaller than 25 ms. The protein has thus been used as a test system to challenge the present paramagnetic NMR methodology both in achieving an extended assignment and in obtaining a suitable number of constraints. 79% of protein protons have been assigned. Analogy with other ferredoxins of known structure has been of help to speed up the final stages of the assignment, although we have shown that this independent information is not necessary. In addition to dipolar connectivities, partially detected through tailored experiments, 3JHN-H alpha, H-bond constraints and dihedral angle constraints on the Cys chi 2 angles have been generated by using a recently derived Karplus-type relationship for the hyperfine shifts of cysteine beta CH2 protons. In total, 456 constraints have been used in distance geometry calculations. The final quality of the structures is satisfactory, with root-mean-square deviation values of 66 pm and 108 pm for backbone and heavy atoms, respectively. The resulting structure is compared with that of Clostridium acidi urici ferredoxin [Duée, E. D., Fanchon, E., Vicat, J., Sieker, L. C., Meyer, J. & Moulis, J.-M. (1994) J. Mol. Biol. 243, 683-695]. The two proteins are very similar in the overall folding, secondary structure elements and side-chain orientations. The C alpha root-mean-square deviation values between the X-ray-determined C. acidi urici ferredoxin structure and the conformer with lowest energy of the C. pasteurianum ferredoxin family is 78 pm (residues 3-53). Discrepancies in residues 26-28 may arise from the disorder observed in the X-ray structure in that region.

Amino Acid Sequence↗
PubMed1995

Mutation of the metal-bridging proton-donor His63 residue in human Cu, Zn superoxide dismutase. Biochemical and biophysical analysis of the His63-->Cys mutant.

The bridging His63 residue in human Cu, Zn superoxide dismutase, which binds both metals, has been replaced by a Cys residue. The mutant protein has been purified from Escherichia coli and appears to be a normal dimer. Spectroscopic techniques (electronic spectroscopies, EPR, nuclear magnetic relaxation dispersion) show that Cys63 binds the zinc ion, but not the copper ion, and that the latter is probably five co-ordinated with three histidine ligands and two water molecules. The reduction potential of the copper ion in the Cu2+/Cu+ pair decreases from 0.41 V to 0.27 V at neutral pH but still remains intermediate between those of the O2/O2- and O2-/H2O2 pairs so that copper can both oxidize and reduce the O2- substrate, a requirement for dismutase activity. The enzyme binds the substrate-analogue azide (N3-), which displaces one water molecule, with near normal affinity, whereas the enzyme activity with the O2- substrate is reduced to less than 1% of wild-type levels at pH 7.8. The properties of the mutant enzyme are discussed in relation to the superoxide-copper electron transfer process and to the catalytic mechanism.

Copper↗
PubMed1995

Three-dimensional solution structure of the oxidized high potential iron-sulfur protein from Chromatium vinosum through NMR. Comparative analysis with the solution structure of the reduced species.

The NMR solution structure of the oxidized HiPIP from Chromatium vinosum has been solved. Despite the fact that the protein is paramagnetic, 85% of the 1H and 80% of the 15N signals have been assigned. Through 1537 NOEs, out of which 1142 were found to be relevant for the structure determination, a family of structures has been obtained by distance geometry calculations. These structures have then been subjected to restrained energy minimization (REM) and restrained molecular dynamics (RMD) calculations in vacuum. Finally, the mean structure of the RMD family has been treated through RMD in water. The RMSD values for the backbone and heavy atoms within the RMD family are 0.57 +/- 0.14 and 1.08 +/- 0.16 A, respectively. These values together with other parameters indicate that the structure is of good quality and as good as the structure of the reduced protein. The RMDw structures of the reduced and oxidized proteins are different beyond the experimental indetermination. The set of constraints for the reduced and oxidized forms have been used to treat the available X-ray structure by RMD in water. The two structures generated in this way are quite similar to their respective solution structures, thus confirming that the experimental constraints are capable of yielding two different structures from the same starting structural model. This is the first time that independently determined solution structures of two redox states of a paramagnetic protein are available. Differences between them and the X-ray structure are discussed.

Amino Acid Sequence↗
PubMed1995

Determination of the [Fe4S4]Cys4 cluster geometry of Desulfovibrio africanus ferredoxin I by 1H NMR spectroscopy.

1D and 2D 1H NMR studies of the Fe4S4 cluster containing ferredoxin I from Desulfovibrio africanus have been carried out with the aim of determining the geometry of the cluster linkages with the 4 Cys side chains that bind the cluster. This required the Cys beta CH resonances of the oxidised protein to be sequence-specifically and stereo-specifically assigned, and this was accomplished by a combination of TOCSY and NOE measurements, allied to model building based on X-ray structures of related ferredoxins. An analysis of the estimated hyperfine shifts of the Cys beta CH resonances with a Karplus-type equation relating the shifts to iron-sulfur-beta carbon-beta proton dihedral angles, taken together with the relative relaxation rates of the two beta CH2 resonances, estimated from their linewidths, then allowed the iron-sulfur-beta-carbon-alpha-carbon dihedral angles to be determined. A novel representation of the NMR data is presented which shows that the cluster dihedral angles are uniquely determined by the NMR data. The analysis reveals that the dihedral angles for D. africanus ferredoxin I are similar to the corresponding angles of other ferredoxins even though there are differences in their 1H NMR spectra. The sequence-specific and stereospecific assignments have been extended by analogy to the related Fe4S4-containing D. gigas ferredoxin I, and the stereospecific assignments to the Fe4S4-containing Thermococcus litoralis ferredoxin.

Chemical Phenomena↗
PubMed1995

The three-dimensional solution structure of the reduced high-potential iron-sulfur protein from Chromatium vinosum through NMR.

The 1H NMR assignment of the reduced HiPIP from Chromatium vinosum available in the literature [Gaillard, J., Albrand, J.-P., Moulis, J.-M., & Wemmer, D. E. (1992) Biochemistry 31, 5632-5639] has been extended up to 85% of the total protein protons. Ninety percent of the nitrogens have been assigned. Then the solution structure has been obtained using as many as 1147 meaningful NOE connectivities. The protein is sizably paramagnetic even though the ground state is a singlet. Nevertheless, the final RMSD values are 0.62 and 1.19 A for the backbone and the heavy atoms, respectively. These values compare well with those for diamagnetic proteins of the same size. The solution structure is discussed in the light of the available structural information from X-ray data.

Amino Acid Sequence↗
PubMed1995

pH-dependent equilibria of yeast Met80Ala-iso-1-cytochrome c probed by NMR spectroscopy: a comparison with the wild-type protein.

BACKGROUND: Cytochrome c has five distinct pH-dependent conformational states, including two alkaline forms of unknown structure. It is believed that in both of the alkaline forms a Lys residue is ligated to the heme, but the identity of the Lys residue is different. Exchange between these forms would require extensive structural rearrangement. Mutation of the heme axial ligand (Met80) to Ala in Saccharomyces cerevisiae iso-1-cytochrome c yields a protein (Ala80cyt c) capable of binding exogenous ligands such as dioxygen and cyanide. We have analyzed the 1H NMR spectra of this mutant at various pH values in the hope of gaining insight into the structure of the acidic and alkaline forms of native cytochrome c. RESULTS: The pH dependence of the 1H NMR spectrum of ferriAla80cyt c is consistent with the high-spin/low-spin transition (pKa = 6.5) observed by absorption spectroscopy. The T1 values for the low-spin form are consistent with OH ligation, as inferred previously from absorption and electron paramagnetic resonance spectroscopic results. The pH-dependent equilibria of ferriAla80cyt c differ from those of the wild-type protein. Both Ala80 and wild-type ferricyt c appear to have the same iron coordination at low pH (approximately equal to 2), while only one alkaline form of Ala80cyt c (versus two for WTcyt c) was detected. CONCLUSIONS: The differences between the pH dependence of the 1H NMR spectra of Ala80cyt c and those of the wild-type protein demonstrate that the heme axial ligands influence the relative energies of the conformational states of cytochrome c. The results are consistent with the notion that a large rearrangement is required to switch between the two alkaline forms.

Cytochrome c Group↗
PubMed1995

[Effects of prolonged administration of branched-chain amino acids on body composition and physical fitness].

No conclusive data are reported about the effects of long-term branched-chain amino-acids (BCAA) administration on metabolic and respiratory parameters during the sustained hand-grip test (SHGT). Bedside, few data are reported about the relationship between BCAA administration and body composition. Thus, BCAA (14 g/die; 50% L-Leucine, 25% L-Isoleucine, 25% L-Valine) were administered orally to 10 untrained male healthy subjects for 30 days. Before (T0) and after (T1) BCAA administration, oxygen uptake (VO2) and pulmonary ventilation (VE) were evaluated at rest ("baseline", B) and during SHGT. Body height and body weight, body mass index (BMI), fat mass (FM), fat-free mass (FFM), arm muscle area (AMA) and maximal voluntary contraction of forearm muscler (GS, grip strength) were also evaluated at T0 and T1. At T1 FFM increased significantly (T0: 62.3 +/- 7.6 kg; T1: 63.1 +/- 7.5 kg; mean +/- SD; p < 0.05), as well as GS (T0: 46.8 +/- 7.9; T1: 50.6 +/- 7.8; p < 0.01). At T0, VO2 increased significantly during the test (B: 271 +/- 36 ml/min; SHGT: 332 +/- 71 ml/min; p < 0.01), whereas at T1 no significant increase was observed (B: 271 +/- 50 ml/min; SHGT: 286 +/- 60 ml/min). We conclude that the physical fitness of BCAA treated subjects was improved, because of the decrease of O2 uptake during SHGT. Moreover, in our subjects, BCAA seemed to promote FFM-protein synthesis mostly.

Adult↗
PubMed1994

Sequence-specific assignment of the 1H and 15N nuclear magnetic resonance spectra of the reduced recombinant high-potential iron-sulfur protein I from Ectothiorhodospira halophila.

A 1H and 15N NMR investigation through two-dimensional and three-dimensional spectroscopy has been performed on the reduced form ([Fe4S4]2+) of the recombinant high-potential iron-sulfur protein (HiPIP) I from Ectothiorhodospira halophila expressed in Escherichia coli. [Fe4S4]2+ clusters in proteins are paramagnetic with a relatively low mu eff of about 0.8 mu B/iron ion, but the paramagnetic effects on nuclear relaxation are so strong as to yield T1 values of a few milliseconds and linewidths of hundreds of hertz for the nuclei closet to the paramagnetic center. Despite these features, 71 out of 73 residues were identified, most of which were assigned completely as far as proton resonances are concerned; as many as 68 residues could be assigned without any reference to the existing X-ray structure. A total of 88% of all protein protons and 58 out of 69 peptide HN nitrogen signals were assigned. To the best of our knowledge, this is the most extensive 1H assignment of a paramagnetic protein to date. Protons sensitive to the proximity of the cluster were assigned through suitable NOE spectroscopy experiments. Three out of the four coordinated cysteines were assigned, and two residues have been identified whose peptide HN protons give rise to H bonds with coordinated sulfur atoms. The inter-residue NOE cross peaks are in qualitative agreement with the secondary and tertiary structure as obtained from the available X-ray crystallographic analysis of the wild-type protein at 250-pm resolution. It is therefore shown that the expressed protein is properly folded and that it is a reliable model for the wild-type protein. These data are meaningful for the detection of structural differences among mutants in future studies.

Amino Acid Sequence↗
PubMed1994

The three-dimensional structure in solution of the paramagnetic high-potential iron-sulfur protein I from Ectothiorhodospira halophila through nuclear magnetic resonance.

The three-dimensional structure in solution of reduced recombinant high-potential iron-sulfur protein iso-I from Ectothiorhodospira halophila was determined using 948 relevant interproton NOEs out of the 1246 observed NOEs. The determination was accomplished using the XEASY program for spectral analysis and the distance geometry (DG) program DIANA for generation of the structure as described by Wüthrich [Wüthrich, K. (1989) Acc. Chem. Res. 22, 36-44]. The FeS cluster was simulated using an amino acid residue constructed for the present work from a cysteinyl residue with an iron and a sulfur atom attached to the terminal thiol. The family of structures obtained from distance geometry were subjected to energy minimization and molecular dynamics simulations using previously defined force field parameters. The quality of these structures at each stage of the refinement process is discussed with respect to the dihedral angle order parameter and the root-mean-square deviation of the atomic coordinates. The latter values for the backbone atoms vary from 67 pm for the distance-geometry structures to 60 pm for the energy-minimized structures to 51 pm for the structures subjected to restrained molecular dynamics. Finally, the structure in best agreement with the NOE constraints has been further treated with extensive restrained molecular dynamics in water. The solution structure is well defined and is very similar to the available X-ray structure. We do not know of any previous determination of the structure of a paramagnetic protein in solution by NMR. The effect of paramagnetism on the quality of the structure determination is discussed.

Bacteria↗