Biomedical subjects

I Bertini

Publications and source records attributed to I Bertini.

At least 91 records · Page 5Linked to original sources

PubMed1998

Off-resonance experiments and contrast agents to improve magnetic resonance imaging.

The effect of off-resonance irradiation on the water proton NMR signal intensity has been investigated as follows: (a) in the presence of a paramagnetic probe like manganese(II); (b) in the presence of bovine serum albumin (BSA) and two gadolinium(III) complexes, Gd-DTPA and Gd-BOPTA; (c) in the presence of cross-linked BSA and the two above-mentioned gadolinium(III) complexes. The experimental data have been rationalized on the basis of the available theoretical models. The effectiveness of the two complexes as contrast agents for MRI has been predicted. It is shown that contrast agents providing comparable longitudinal and transverse relaxation rate enhancements are those of general interest for off-resonance magnetization transfer-MRI.

Animals↗

PubMed1998

Nuclear and Electron Relaxation in Magnetic Exchange Coupled Dimers: Implications for NMR Spectroscopy

The transition probabilities and the lifetimes have been calculated for the levels arising from magnetic exchange coupling in the following electron spin pairs: SA = ½-SB = ½; SA = ½-SB = 1; SA = ½-SB = 32; SA = 1-SB = 1. Such transition probabilities and lifetimes have been expressed as a function of the relaxation properties of the uncoupled spins in the assumption that magnetic coupling does not provide further relaxation pathways, and that the coupling frequency is large with respect to the electron relaxation rates of both spins. From the above values, nuclear relaxation as a function of the intensity of the external magnetic field has been calculated for nuclei dipole-coupled with either electron spin. The calculated nuclear relaxation dispersion has been then analyzed in terms of an "effective" electron relaxation time, the knowledge of which is important for NMR of magnetic coupled systems. The calculations provide a basis for understanding electron relaxation in magnetic-coupled dimers. Comparison with available experimental literature data is presented. Copyright 1998 Academic Press. Copyright 1998 Academic Press

Journal Article↗

PubMed1998

High magnetic field consequences on the NMR hyperfine shifts in solution

Pseudocontact shifts arise from the isotropic reorientational average of the dipolar coupling between unpaired electron and nuclei, in the presence of magnetic susceptibility anisotropy. The effect of residual orientation due to high magnetic fields on pseudocontact shifts is evaluated here. The effect is found to be smaller and of opposite sign with respect to another novel effect of high magnetic fields on hyperfine shifts due to saturation of the electron spin magnetic moment as described by the Brillouin equation. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

Comparison of different techniques to measure body composition in moderately active adolescents.

OBJECTIVE: To evaluate the differences in the estimate of body fat percentage (%FM) and the amount (kg) of fat free mass (FFM) by different methods in 26 moderately active adolescents very similar in age, body fatness, and training status. METHODS: Mean (SD) age was 16.7 (0.9) years, height was 177.0 (5.1) cm, and weight 68.0 (5.2) kg. %FM was assessed using dual-energy x ray absorptiometry (DXA) and two skinfold prediction equations: that of Slaughter et al (%FM Sla) and that of Deurenberg et al (%FM Deu). In the same way, FFM was measured using DXA and different impedance equations: those of Suprasongsin et al (FFM Sup), Schaefer et al (FFM Sch), Houtkooper et al (FFM Hou), and Deurenberg et al (FFM Deu). To determine the interchangeability of the different methods of measuring %FM and FFM, one way analysis of variance, standard error (SE), and coefficient of variation (CV%) ((SD/mean) x 100) were used. RESULTS: On average, no significant statistical differences were observed between the values determined for %FM: DXA value, 11.7 (5.4%); %FM Sla, 10.9 (4.0)%; %FM Deu, 11.5 (2.3)%. On the other hand, SE and CV% between each pair of the three methods used showed very large variability. With regard to the measurement or prediction of FFM, the mean value measured by DXA was significantly higher than that predicted by the equation of Sch (+7.2 kg, p < 0.001), Deu (+3.2 kg, p < 0.001), and Hou (+2.6 kg, p < 0.001), whereas it was lower than that predicted by the equation of Sup (-1.6 kg, p < 0.05). The Hou and Deu values were the only two that, on average, did not differ in a statistically significant way, although they showed the highest CV%. CONCLUSIONS: In our sample of moderately active adolescents the estimated values for %FM and FFM appear to be highly dependent on method.

Absorptiometry, Photon↗

PubMed1997

Characterization of the unbound 2[Fe4S4]-ferredoxin-like photosystem I subunit PsaC from the Cyanobacterium synechococcus elongatus.

Recombinant PsaC was reconstituted in vitro and investigated by UV/vis, EPR, and 1H NMR spectroscopy. Its UV/vis and EPR spectroscopic properties correspond to those of the wild-type protein. Fast repetition 1D and 2D 1H NMR spectra allowed the sequence-specific assignment of the hyperfine-shifted proton resonances of the cluster-ligating resonances, taking advantage also of chemical shift analogies with other 4 and 8 Fe ferredoxins and a structural model for PsaC. The Calpha-Cbeta-S-Fe dihedral angles of the cluster ligands could be estimated from the chemical shifts and relaxation properties of their betaCH2 protons. All NMR-derived structural information on PsaC confirms its similarity to smaller 8Fe ferredoxins serving as electron transfer proteins in solution. Partial reduction of PsaC leads to an intermediate species with strongly exchange broadened 1H NMR resonances. The intermolecular electron exchange rate is estimated to be in the 10(2)-10(4) s-1 range, the intramolecular electron exchange rate between the two [Fe4S4] clusters to be higher than 10(4) s-1. The consequences of these findings for the electron transfer in photosystem I are discussed.

Cyanobacteria↗

PubMed1997

Solution structure of reduced microsomal rat cytochrome b5.

The solution structure of the major form of the reduced soluble fragment of rat microsomal cytochrome b5 has been solved through 1H-NMR spectroscopy. The protein contains 98 amino acids. Proton assignment was available for residues 1-94, except 90 [Guiles, R. D., Basus, V. J., Kuntz, I. D. & Waskell, L. (1992) Biochemistry 31, 11,365-11,375] and has been confirmed. From 1722 NOEs, of which 1203 were found to be meaningful, a family of 40 energy-minimized structures has been obtained with average backbone rmsd (for residues 5-89) of 0.078 +/- 0.018 nm and average target function of 0.0045 nm2, no distance violations being larger than 0.029 nm. The structure has been compared with the X-ray structure of the oxidized rat mitochondrial isoenzyme and with that of the highly similar bovine microsomal isoenzyme in the oxidized form. The analysis of the elements of secondary structure is instructive in terms of their stability and of their occurrence in related structures, and of the capability of NMR and X-ray spectroscopy to observe them. Some detailed structural variations are noticed among the solved structures of the various isoenzymes and between solid and solution. The structural features in solution of the residues proposed to be involved in protein-protein recognition are found to be largely conserved with respect to the solid state.

Amino Acid Sequence↗

PubMed1997

Solution structure of the paramagnetic complex of the N-terminal domain of calmodulin with two Ce3+ ions by 1H NMR.

The solution structure of the dicerium(III) complex of the N-terminal domain of calmodulin (Ce2-TR1C hereafter) has been solved employing paramagnetic T1 relaxation enhancements and pseudocontact shifts introduced by the Ce3+ ions, together with conventional NOE constraints. The use of pseudocontact shift constraints constitutes the first attempt to locate metal ions within a protein structure by NMR. Like calcium(II), paramagnetic cerium(III) has been found to bind to the two metal binding sites of the TR1C fragment of calmodulin in a cooperative manner. Due to the presence of pseudocontact interactions between the Ce3+ ions and protons of the 76-residue protein, the 1H NMR spectra of the complex show resonances shifted between +22 and -9 ppm. Eighty percent of its proton resonances could be assigned through a standard approach using TOCSY/COSY and NOESY spectra and through 1D NOE difference spectra for the broad resonances of protons close to the paramagnetic ions. A family of structures was calculated by means of the torsion angle dynamics program DYANA [Güntert, P., Mumenthaler, C., & Wüthrich, K. (1996) XVIIthInternational Conference on Magnetic Resonance inBiological Systems (Abstract)] using 1012 NOEs. Longitudinal proton relaxation times helped to roughly define the position of the metal ions within the protein. A total of 381 pseudocontact shift constraints, whose evaluation and use are critically discussed, have then been added to further refine the metal coordinates within the protein frame and to improve the structure resolution. A dramatic resolution improvement of the metal coordinates together with a sizable resolution improvement in the regions close to the paramagnetic centers, where the number of NOEs is low, is observed. The good quality of the solution structure permitted a meaningful comparison with the solid-state structure of calcium-loaded calmodulin at 1.7 A resolution [Chattopadhyaya, R., Meador, W. E., Means, A. R., & Quiocho, F. A. (1992) J. Mol. Biol. 228, 1177]. The Ce2-TR1C complex is overall more compact than the Ca form.

Amino Acid Sequence↗

PubMed1997

ePHOGSY experiments on a paramagnetic protein: location of the catalytic water molecule in the heme crevice of the oxidized form of horse heart cytochrome c.

The hydration properties of the oxidized form of horse heart cytochrome c have been studied by 1H NMR spectroscopy. Application of ePHOGSY (enhanced protein hydration observed through gradient spectroscopy) experiments over a paramagnetic molecule provided firm spectroscopic evidence of the presence of a water molecule in the heme crevice. A few intermolecular NOEs have been used to locate the water molecule at about 0.65 nm away from the iron atom and to compare the position observed in solution with that observed in the crystal structure and in solution for the reduced state. The resulting picture is that there is a detectable movement of the water molecule upon oxidation.

Animals↗

PubMed1997

Solution structure of oxidized horse heart cytochrome c.

The solution structure of oxidized horse heart cytochrome c was obtained at pH 7.0 in 100 mM phosphate buffer from 2278 NOEs and 241 pseudocontact shift constraints. The final structure was refined through restrained energy minimization. A 35-member family, with RMSD values with respect to the average structure of 0.70 +/- 0.11 A and 1.21 +/- 0.14 A for the backbone and all heavy atoms, respectively, and with an average penalty function of 130 +/- 4.0 kJ/mol and 84 +/- 3.7 kJ/mol for NOE and pseudocontact shift constraints, respectively (corresponding to a target function of 0.9 A2 and 0.2 A2), was obtained. The solution structure is somewhat different from that recently reported (Qi et al., 1996) and appears to be similar to the X-ray structure of the same oxidation state (Bushnell et al., 1990). A noticeable difference is a rotation of 17 +/- 8 degrees of the imidazole plane between solid and solution structure. Detailed and accurate structural determinations are important within the frame of the current debate of the structural rearrangements occurring upon oxidation or reduction. From the obtained magnetic susceptibility tensor a separation of the hyperfine shifts into their contact and pseudocontact contributions is derived and compared to that of the analogous isoenzyme from S. cerevisiae and to previous results.

Amino Acid Sequence↗

PubMed1997

Characterization of a partially unfolded high potential iron protein.

A partially unfolded state of the Fe4S4-containing high potential iron-sulfur protein from Chromatium vinosum has been detected and characterized by NMR spectroscopy following addition of a concentrated solution of guanidinium chloride to the native protein. This intermediate species (i) maintains the polymetallic center, (ii) exhibits a largely collapsed secondary structure, and (iii) undergoes fast cluster decomposition upon oxidation. This information is framed into the knowledge about this class of proteins, and the possible role of this intermediate with respect to the in vivo folding/unfolding process is discussed as well its role in the slow hydrolytic degradation characteristic of oxidized HiPIPs.

Chromatium↗

PubMed1997

The D13C variant of Bacillus schlegelii 7Fe ferredoxin is an 8Fe ferredoxin as revealed by 1H-NMR spectroscopy.

The N-terminal cluster binding motif Cys8XXXXXXXCys16....Cys49 of Bacillus schlegelii 7Fe ferredoxin, which provides the ligands to the [Fe3S4]+ cluster, was modified by the mutation Asp13 --> Cys. The mutant D13C is expressed in Escherichia coli as an 8Fe ferredoxin, with NMR properties similar to those of clostridial-type ferredoxins. The full assignment of the hyperfine shifted resonances indicates that Cys13 serves as ligand to the new fourth iron atom in the N-terminal cluster despite the atypical binding sequence CysXXXXCysXXCys....Cys. The C alpha-C beta-S-Fe dihedral angles of all cysteine ligands to the two [Fe4S4]2+ clusters of the D13C variant are similar to those observed in other 8Fe and 4Fe ferredoxins.

Aspartic Acid↗

PubMed1997

Solution structure of oxidized Saccharomyces cerevisiae iso-1-cytochrome c.

The solution structure of oxidized Saccharomycescerevisiae Cys102Ser iso-1-cytochromechas been determined using 1361 meaningful NOEs (of 1676 total) after extending the published proton assignment [Gao, Y., et al. (1990) Biochemistry 29, 6994-7003] to 77% of all proton resonances. The NOE patterns indicate that secondary structure elements are maintained upon oxidation in solution with respect to the solid state and solution structures of the reduced species. Constraints derived from the pseudocontact shifts [diamagnetic reference shift values are those of the reduced protein [Baistrocchi, P., et al. (1996) Biochemistry 35, 13788-13796]] were used in the final stages of structure calculations. After restrained energy minimization with constraints from NOEs and pseudocontact shifts, a family of 20 structures with rmsd values of 0.58 +/- 0.08 and 1.05 +/- 0.10 A (relative to the average structure) for the backbone and all heavy atoms, respectively, was obtained. The solution structure is compared with the crystal structure and the structures of related systems. Twenty-six amide protons were detected in the NMR spectrum 6 days after the oxidized lyophilized protein was dissolved in D2O (pH 7.0 and 303 K); in an analogous experiment, 47 protons were observed in the spectrum of the reduced protein. The decrease in the number of nonexchanging amide protons, which mainly are found in the loop regions 14-26 and 75-82, confirms the greater flexibility of the structure of oxidized cytochrome c in solution. Our finding of increased solvent accessibility in these loop regions is consistent with proposals that an early step in unfolding the oxidized protein is the opening of the 70-85 loop coupled with dissociation of the Met80-iron bond.

Amino Acid Sequence↗

PubMed1997

An NMR study of the 7Fe-8S ferredoxin from Rhodopseudomonas palustris and reinterpretation of data on similar systems.

The oxidized 7Fe-8S ferredoxin from Rhodopseudomonas palustris is shown to possess a unique 1H NMR spectrum displaying at least one hyperfine-shifted beta-CH2 signal for each cysteine bound to the [3Fe-4S] cluster. COSY and TOCSY spectra and 1- and 2-dimensional NOE experiments, in conjunction with a thorough reexamination of the 1H NMR data on similar systems, permitted the sequential assignment of all of the cysteine beta-CH2 protons even in the absence of the amino acid sequence. The sequential assignment stems on the homology of the hyperfine shift pattern with those of other sequenced 7Fe-8S ferredoxins, which points to a substantial homology in tertiary structure. From the assignment, an analysis of the antiferromagnetic coupling in the [3Fe-4S] system was performed on the basis of a general model of exchange coupling. The NMR signal patterns of [3Fe-4S] clusters in both 3Fe-4S and 7Fe-8S ferredoxins have been discussed, and some correlations are proposed between signal patterns and the primary sequence.

Cysteine↗

PubMed1997

Pseudocontact shifts as constraints for energy minimization and molecular dynamics calculations on solution structures of paramagnetic metalloproteins.

The pseudocontact shifts of NMR signals, which arise from the magnetic susceptibility anisotropy of paramagnetic molecules, have been used as structural constraints under the form of a pseudopotential in the SANDER module of the AMBER 4.1 molecular dynamics software package. With this procedure, restrained energy minimization (REM) and restrained molecular dynamics (RMD) calculations can be performed on structural models by using pseudocontact shifts. The structure of the cyanide adduct of the Met80Ala mutant of the yeast iso-1-cytochrome c has been used for successfully testing the calculations. For this protein, a family of structures is available, which was obtained by using NOE and pseudocontact shifts as constraints in a distance geometry program. The structures obtained by REM and RMD calculations with the inclusion of pseudocontact shifts are analyzed.

Algorithms↗

PubMed1997

Fatty acid composition of human milk in Italy.

The fatty acid composition of breast milk from 20 Italian women, delivering at term and on ad libitum diets, was analyzed with high-resolution gas chromatography. Milk samples were collected twice a day, on the 1st, 4th, 7th, 14th, 21st and 28th day after colostrum appearance. No significant differences were detected between the two daily samples. During the maturation process a significant reduction in long-chain polyunsaturated fatty acids of the n-6 series (p = 0.002) and n-3 series (p = 0.005) was recorded, particularly in arachidonic acid (p = 0.035), docosatetraenoic acid (p = 0.035) and docosahexaenoic acid (p = 0.032). The linoleic acid/n-6 and alpha-linolenic acid/n-3 ratios increased (p = 0.024 and p = 0.037), while the docosatetraenoic/docosahexaenoic acid ratio decreased (p = 0.032). The fatty acid composition of mature milk was the following: saturated 45.50%; unsaturated 54.51%; monounsaturated 42.69%; polyunsaturated 11.82%; long-chain polyunsaturated 1.27%; linoleic acid 9.79%, and alpha-linolenic acid 0.36%. The fatty acid composition of milk collected from Italian women appears similar to that of women in other southern European countries and, therefore, could reflect dietary habits.

Adult↗

PubMed1997

Paramagnetic relaxation as a tool for solution structure determination: Clostridium pasteurianum ferredoxin as an example.

The possibility of using the relaxation properties of nuclei for solution structure determination of paramagnetic metalloproteins is critically evaluated. First of all, it is theoretically and experimentally demonstrated that magnetization recovery in nonselective inversion recovery experiments can be approximated to an exponential in both diamagnetic and paramagnetic systems. This permits the estimate of the contribution of paramagnetic relaxation when dominant or sizable. Then, it is shown that the averaging of paramagnetic relaxation rates due to cross relaxation is often tolerably small with respect to the use of paramagnetic relaxation rates as constraints for structural determination. Finally, a protocol is proposed to use such paramagnetic relaxation rates, which depend on the sixth power of the metal to resonating nucleus distance, as constraints for solution structure determination of proteins. As an example, the available solution structure of the oxidized ferredoxin from Clostridium pasteurianum has been significantly improved in resolution especially in the proximity of the metal ions by using 69 new constraints based on paramagnetic relaxation.

Clostridium↗

PubMed1996

NMR characterization and solution structure determination of the oxidized cytochrome c7 from Desulfuromonas acetoxidans.

The solution structure of the three-heme electron transfer protein cytochrome c7 from Desulfuromonas acetoxidans is reported. The determination of the structure is obtained through NMR spectroscopy on the fully oxidized, paramagnetic form. The richness of structural motifs and the presence of three prosthetic groups in a protein of 68 residues is discussed in comparison with the four-heme cytochromes c3 already characterized through x-ray crystallography. In particular, the orientation of the three hemes present in cytochrome c7 is similar to that of three out of four hemes of cytochromes c3. The reduction potentials of the individual hemes, which have been obtained through the sequence-specific assignment of the heme resonances, are discussed with respect to the properties of the protein matrix. This information is relevant for any attempt to understand the electron transfer pathway.

Amino Acid Sequence↗

PubMed1996

Three-dimensional solution structure of Saccharomyces cerevisiae reduced iso-1-cytochrome c.

Two-dimensional 1H NMR spectra of Saccharomyces cerevisiae reduced iso-1-cytochrome c have been used to confirm and slightly extend the assignment available in the literature. 1702 NOESY cross-peaks have been assigned, and their intensities have been measured. Through the program DIANA and related protocols (Güntert, 1992), a solution structure has been obtained by using 1442 meaningful NOEs and 13 hydrogen-bond constraints. The RMSD values with respect to the mean structure for the backbone and all heavy atoms for a family of 20 structures are 0.61 +/- 0.09 and 0.98 +/- 0.09 A, the average target function value being as small as 0.57 A2. The larger number of slowly exchanging amide NHs observed in this system compared to that observed in the cyanide derivative of oxidized Ala 80 cytochrome c suggests that the oxidized form is much more flexible and that the backbone protons are more solvent accessible. Comparison of the present structure with the crystal structures of reduced yeast cytochrome c and of the complex between cytochrome c peroxidase and oxidized yeast cytochrome c reveals substantial similarity among the backbone conformations but differences in the residues located in the region of protein-protein interaction. Interestingly, in solution the peripheral residues involved in the interaction with cytochrome c peroxidase are on average closer to the position found in the crystal structure of the complex than to the solid state structure of the isolated reduced from.

Amino Acid Sequence↗