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M Paci

Publications and source records attributed to M Paci.

At least 73 records · Page 4Linked to original sources
PubMed1990

Structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae.

The covalent structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae isolates, pathogenic on various species of citrus trees, has been deduced from 1D and 2D 1H- and 13C-NMR spectra combined with extensive FAB-MS data and results of some chemical reactions. Similarly to syringomicins and syringostatins, produced by other plant pathogenic strains of P. syringae pv. syringae, syringotoxin is a lipodepsinonapeptide. Its peptide moiety corresponds to Ser-Dab-Gly-Hse-Orn-aThr-Dhb-(3-OH)Asp-(4-Cl)Thr with the terminal carboxy group closing a macrocyclic ring on the OH group of the N-terminal Ser, which in turn is N-acetylated by 3-hydroxytetradecanoic acid.

Amino Acid Sequence↗
PubMed1990

Reconstitution of Cu,Zn-superoxide dismutase by the Cu(I).glutathione complex.

The reconstitution of Cu,Zn-superoxide dismutase from the copper-free protein by the Cu(I).GSH complex was monitored by: (a) EPR and optical spectroscopy upon reoxidation of the enzyme-bound copper; (b) NMR spectroscopy following the broadening of the resonances of the Cu(I).GSH complex after addition of Cu-free,Zn-superoxide dismutase; and (c) NMR spectroscopy of the Cu-free,Co(II) enzyme following the appearance of the isotropically shifted resonances of the Cu(I), Co enzyme, Cu(I).GSH was found to be a very stable complex in the presence of oxygen and a more efficient copper donor to the copper-free enzyme than other low molecular weight Cu(II) complexes. In particular, 100% reconstitution was obtained with stoichiometric copper at any GSH:copper ratio between 2 and 500. Evidence was obtained for the occurrence of a Cu(I).GSH.protein intermediate in the reconstitution process. In view of the inability of copper-thionein to reconstitute Cu,Zn-superoxide dismutase and of the detection of copper.GSH complexes in copper-over-loaded hepatoma cells (Freedman, J.H., Ciriolo, M.R., and Peisach, J. (1989) J. Biol. Chem. 264, 5598-5605), Cu(I).GSH is proposed as a likely candidate for copper donation to Cu-free,Zn-superoxide dismutase in vivo.

Animals↗
PubMed1990

Assignment of imidazole resonances from two-dimensional proton NMR spectra of bovine Cu,Zn superoxide dismutase. Evidence for similar active site conformation in the oxidized and reduced enzyme.

Two-dimensional 1H-NMR spectra were carried out on bovine Cu(I),Zn superoxide dismutase. The ring protons of the single tyrosine and of the 4 phenylalanines were identified from COSY spectra. From NOESY spectra all imidazole C-resonances could be specifically assigned to each of the 8 histidines using the crystal coordinates of the Cu(II),Zn enzyme. Since 6 histidines are involved in the structure of the active site, this result implies nearly identical active site conformations for the two oxidation states of the catalytic cycle of this enzyme, in line with its diffusion-limited rate.

Animals↗
PubMed1990

Mapping the copper ligands of Cu,Zn superoxide dismutase by nuclear Overhauser enhancement of the isotropically shifted 1H-NMR lines of the Cu,Co derivative.

Transient nuclear Overhauser effect (NOE) experiments were made with bovine Cu,Co superoxide dismutase on the hyperfine shifted resonances of protons of the imidazole groups bound to the Cu,Zn cluster of the native enzyme. Assignments of lines based on the observed magnetization transfers were satisfactorily obtained only by altering the arrangement of the ligands around the catalytically active copper shown by X-ray crystallography.

Animals↗
PubMed1990

A study of the intracellular effects of glutathione by 1H-spin echo NMR of intact human erythrocytes.

The effect of adding either reduced (GSH) or oxidized (GSSG) glutathione to intact human erythrocytes was investigated by 1H-spin echo NMR, which allows direct observation of relatively concentrated low molecular weight compounds within intact cells. A specific region of the spectrum was affected by addition of GSH, with the appearance of new peaks that were diagnostic of an increase of intracellular GSH. These changes did not occur in hemolysates, and did not involve extra-cytosol GSH either free or membrane-bound. These results indicate that the intracellular redox balance of glutathione is shifted toward the reduced state by exogenous glutathione, possibly via a signal transferring system of the cell membrane.

Erythrocytes↗
PubMed1988

Structure-function relationship in Escherichia coli translational initiation factors. Characterization of IF1 by high-resolution 1H-NMR spectroscopy.

Escherichia coli translational initiation factor IF1 was studied by 1H-NMR spectroscopy at 400 MHz. IF1 displays a very well resolved spectrum in both aromatic and aliphatic regions. Other spectral characteristics include relatively narrow resonance lines and lack of relevant cross-relaxation phenomena. The resonances of the aromatic residues, in particular of the two His and two Tyr, were assigned by selective chemical modifications and spectroscopic techniques to individual residues in the protein sequence. The relative mobility of various residues of IF1 has been evaluated on the basis of the spin-lattice relaxation times which are rather short and homogeneous. Overall the factor appears to have a complex secondary and tertiary structure and to be a flexible protein whose residues have a high degree of internal mobility.

Escherichia coli↗
PubMed1988

Cyanide binding to Cu, Zn superoxide dismutase. An NMR study of the Cu(II), Co(II) derivative.

The Cu,Co superoxide dismutase derivative, in which the native Zn(II) was replaced by Co(II), was investigated by 1H NMR spectroscopy at pH 7.0 in the presence of CN- and N-3. Addition of either anion produced large but remarkably different variations in the position of the histidine proton signals bound to the metal cluster. The resonances of the histidines bound to the copper broadened at low CN- concentrations (6 X10(-5)-16.5 X 10(-3) M KCN, in the presence of 1.5 mM protein) and narrowed again, with changed chemical shifts at [KCN] greater than 10(-2) M. At 7 degrees C two resonances split into two pairs of lines as a function of [CN-]. The temperature dependence of these resonances, in the presence of nonsaturating [CN-], suggests a slow exchange between two forms of the protein-bound copper in the presence of the anion. The apparent activation parameters associated with the interconversion of the two species indicate a local conformational change in the presence of CN-. No evidence of temperature dependence was seen in the spectrum in the presence of N-3, which, on the other hand, was fully removed from the copper by addition of CN-. No evidence was obtained for removal by CN- of a histidine bound to the copper as previously reported for low affinity anions at pH 5.5 (Bertini, I., Lanini, G., Luchinat, C., Messori, L., Monanni, R., and Scozzafava, A. (1985) J. Am. Chem. Soc. 107, 4391-4396). These results indicate that CN- has a unique pattern of binding to the enzyme copper. Since catalytic and structural data indicate that CN- is the only appropriate substrate analogue for the Cu,Zn superoxide dismutase, data from anions with much less affinity may lead to misleading conclusions on the mechanism of anion and substrate binding to the enzyme.

Animals↗
PubMed1988

Differential binding of anions to the active site of Cu,Zn superoxide dismutase. A study of the Co,Zn enzyme derivative.

The reaction of N3- with Co,Zn superoxide dismutase, a good analogue of the native Cu,Zn enzyme, was studied in the presence and absence of phosphate, which is known to perturb the spectroscopic properties of the cobalt chromophore in the Co,Zn enzyme. EPR, NMR, and optical titrations demonstrated the formation of different adducts for N3- depending on the presence of phosphate, at variance with results previously obtained with CN- [3]. This evidence indicates that the mechanism of anion binding to Cu,Zn superoxide dismutase cannot be described on the basis of data obtained with a single type of anions.

Animals↗
PubMed1988

1H and 31P NMR studies of the binding of low-affinity anions to Cu,Zn superoxide dismutase.

Anions that do not coordinate to the catalytically active copper ion of Cu,Zn superoxide dismutase, but still affect the activity of the enzyme by weaker interactions with the protein moiety surrounding the active site (low affinity anions), uniformly perturbed the 1H NMR line of the NH group of the copper ligand His 46. This effect was detected on the enzyme having Co(II) substituted for the native Zn(II), in which the resonances of residues bound to the copper are detected because of the antiferromagnetic coupling between Cu(II) and Co(II). The interaction with the enzyme of phosphate, a good representative of low-affinity anions, was also studied by 31P NMR of the native enzyme and of enzyme samples covalently modified at all lysines or at the Arg 141, which is 5 A away from the copper. The results obtained indicate that Arg 141 is a likely candidate for binding of low-affinity anions in the vicinity of the copper and that the 1H NMR line of His 46 NH is diagnostic for such an interaction.

Animals↗
PubMed1986

Proteins from the prokaryotic nucleoid: 1H NMR study of the quaternary structure of Escherichia coli DNA binding protein NS (HU).

The quaternary interactions of Escherichia coli DNA binding proteins NS1, NS2, and NS (NS1 + NS2) have been studied by 1H NMR spectroscopy at 400 MHz following the reversible spectral changes produced by temperature increases on the resonances (Phe ring and His C-2 protons) whose spectral characteristics reflect the formation and dissociation of either homologous or heterologous interactions. These changes include (a) a progressive intensity decrease of the Phe resonances shifted to high field by stacking interactions, (b) a progressive intensity increase of the resonances due to freely rotating Phe, and (c) splitting of the His C-2 proton resonance. The association constants and thermodynamic parameters for the homologous and heterologous interactions were calculated from the molar fractions of the relevant molecular species by assuming that the above effects are due to the existence of simple association equilibria. It was found that two (out of three) phenylalanine residues of each polypeptide chain are involved in quaternary interactions. Quantitative data concerning the internal mobility and mutual orientations in aggregates of these Phe rings were also obtained. From the calculated association constants, from comparison of these data with recent protein-protein cross-linking results [Losso, M. A., Pawlik, R. T., Canonaco, M. A., & Gualerzi, C. O. (1986) Eur. J. Biochem. 155, 27-32], and from other considerations, we suggest that even though stacking of the Phe rings occurs at the interface between monomers, the temperature-dependent alteration of the Phe spectrum monitors shifts of the dimer in equilibrium tetramer equilibrium whereas the splitting of the His C-2 proton resonance most likely monitors the equilibrium between tetramers and larger aggregates.

Bacterial Proteins↗
PubMed1985

Structure-function relationship in Escherichia coli initiation factors. Biochemical and biophysical characterization of the interaction between IF-2 and guanosine nucleotides.

Equilibrium dialysis and protection from heat inactivation and proteolysis show that initiation factor 2 (IF-2) interacts not only with GTP but also with GDP and that its conformation is changed upon binding of either nucleotide. The apparent Ka (at 25 degrees C) for the IF-2 X GDP and IF-2 X GTP complexes was 8.0 X 10(4) and 7.0 X 10(3) M(-1), respectively. The lower affinity for GTP is associated with a more negative delta S0. The interaction, monitored by 1HNMR spectroscopy, is characterized by fast exchange and results in line broadening and downfield shift of the purine C-8 and ribose C-1' protons of GTP as well as of the beta, gamma-methylene protons of (beta-gamma-methylene)guanosine 5'-triphosphate. The interaction of guanosine nucleotides with IF-2 requires an H bond donor (or acceptor) group at position C-2 of the purine and involves the beta- and/or gamma-phosphate of the nucleotide while the ribose 2'-OH group or the integrity of the furan ring are less critical. IF-2 binds to ribosomal particles with decreasing affinity: 30 S greater than 70 S greater than 50 S. GTP and GDP have no effect on the binding to 70 S. GTP stimulates the binding to the 30 S and depresses somewhat the binding to the 50 S subunits; GDP has the opposite effect. These results seem to rule out that the release of IF 2 from 70 S is due to a "GDP-conformation" of the factor incompatible with its permanence on the ribosome. The rate and the extent of 30 S initiation complex formation are approximately 2-fold higher with IF-2 X GTP than with IF-2 alone. At low concentrations of IF-2 and 30 S subunits, GDP inhibits this reaction, acting as a strong competitive inhibitor of GTP (Ki = 1.25 X 10(-5)m) and preventing IF-2 from binding to the ribosomal subunit.

Escherichia coli↗
PubMed1985

The interaction between initiation factor 3 and 30 S ribosomal subunits studied by high-resolution 1H NMR spectroscopy.

The interaction between Escherichia coli translational initiation factor 3 (IF-3) (Mr = 20668) and 30 S ribosomal subunits or fragmented 16 S rRNA was followed by 1H NMR spectroscopy. Upon addition of increasing yet largely substoichiometric amounts of deuterated 30 S ribosomal subunits, selective line broadenings and some chemical shift changes were observed. These effects can be fully reversed by increasing the temperature and/or the ionic strength. The selective line broadenings, which are explained by a medium-fast to fast exchange dynamics between free and bound IF-3 with loss of internal mobility of the protons, shed light on the amino acid residues of IF-3 involved in or affected by the binding to the 30 S subunits. Some effects (i.e. implication of 1 tyrosine, 1 phenylalanine, and some arginine and lysine residues) are seen with both 30 S subunits and rRNA while others (i.e. implication of a second tyrosine or phenylalanine residue of a group of hydrophobic residues and, possibly, of the single histidine residue), seen only or preferentially with 30 S subunits, may reflect additional interactions exclusively occurring at the ribosomal level.

Amino Acids↗
PubMed1984

Structure-function relationship in Escherichia coli translational initiation factors. Characterization of IF-3 by high resolution 1H NMR spectroscopy.

Translational initiation factor-3 (IF-3) was characterized by 1H NMR spectroscopy as a function of pH and temperature and following chemical modifications. Spin-lattice relaxation times for individual resonances and bands were also measured. Several resonances were assigned to different amino acid residues by different criteria. Among these are the CH3-N of the N-terminal methionine which appears free, mobile, and very sensitive to the modification of several physicochemical parameters as well as the 3,5 and 2,6 protons of the three tyrosines (two of which play a role in the function of IF-3) which were found to be located in different magnetic environments. Two of these residues appear to be close to each other and in the vicinity of a slow reacting arginine within the tertiary structure of the factor. The properties and the titration behavior of the imidazole proton resonances suggest that the single His residue is partially buried in the protein structure. Characteristic of the IF-3 spectrum also is the presence of an abundant subset of Arg delta-CH2, Lys epsilon-CH2, and CH3 protons displaying clear cut upfield perturbations. These are probably due to the coming together of two or more apolar "fronts" which possibly arise from distant parts of the molecule and result in the close proximity between aromatic rings and aliphatic side chains. The IF-3 spectrum also includes several distinct methyl resonances significantly shifted upfield by aromatic ring currents. Overall, the characteristics of the spectrum, its relative insensitivity to temperature and ionic strength, and the existence of extensive cross-relaxation phenomena indicate that IF-3 has a highly folded tertiary structure with abundant hydrophobic regions. In spite of some heterogeneity in the distribution of the side chain environments, no indication was found for the existence of distinct domains or, at least, of extensive regions with higher mobility.

Bacterial Proteins↗
PubMed1984

Proteins from the prokaryotic nucleoid. High-resolution 1H NMR spectroscopic study of Escherichia coli DNA-binding proteins NS1 and NS2.

The 1H-NMR spectra of the two Escherichia coli basic, low-Mr (approximately equal to 9000) DNA-binding proteins NS1 and NS2 and of their native complex NS were studied at 400 MHz and a number of resonances and resonance peaks were assigned. As in the case of some eukaryotic histones, the presence of a large number of high-field perturbed Phe resonances, several shielded and deshielded methyl resonances and backbone NH protons quite inaccessible to the solvent clearly indicate the existence of extensive tertiary and, even more so, quaternary structures involving hydrophobic interactions. These structures are lost upon heating, but readily reform upon cooling. Spectral differences between NS1, NS2 and NS and the greater thermal stability of NS indicate that molecules of the heterologous subunits (NS1 and NS2) aggregate (dimerize) preferentially in comparison to the self-aggregation of the homologous subunits. Unlike those of the eukaryotic histones, the tertiary and quaternary structures of NS are insensitive to extensive variations of the ionic strength.

Bacterial Proteins↗