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Biomedical subjects

I Bertini

Publications and source records attributed to I Bertini.

At least 19 recordsLinked to original sources
PubMed2004

Crystal structure of the catalytic domain of human matrix metalloproteinase 10.

The catalytic domain of matrix metalloproteinase-10 (MMP-10) has been expressed in Escherichia coli and its crystal structure solved at 2.1 A resolution. The availability of this structure allowed us to critically examine the small differences existing between the catalytic domains of MMP-3 and MMP-10, which show the highest sequence identity among all MMPs. Furthermore, the binding mode of N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid (NNGH), which is one of the most known commercial inhibitors of MMPs, is described for the first time.

Amino Acid Sequence↗
PubMed2003

A follow-up study of the variations in the body composition of karate athletes.

We monitored body composition variations in nine elite karateists during a 3-year period (T(1), T(2), T(3)). Arm circumference (AC) and six skinfolds were measured. Arm muscle circumference (AMC) and area (AMA) were then calculated. Fat mass (FM) was calculated using the Durnin-Womersley, Sloan-Weir (S-W), Katch-McArdle and Lohman equations. The only significant increase regarded the AC, AMC and AMA measurements between T(1) and T(2). The time course of the predicted FM amount using the different equations was the same. In particular, the S-W equation did not seem to reflect the modifications in the amount of FM as evidenced by the total sum of the six skinfolds. Our subjects were characterized by a substantial uniformity in body composition parameters measured in this 3-year follow-up study.

Adult↗
PubMed2003

Anthropometric features and body composition of young athletes practicing karate at a high and medium competitive level.

The aim of the study was to examine the anthropometric features and body composition of athletes practising karate at a high and medium competitive level. Our study was carried out on a sample of 35 subjects practising karate and aged from 16.0 to 32.5 years. This sample was divided into two groups: group 1 ( n=14 elite athletes) and group 2 ( n=21 amateur athletes). Various anthropometric measurements were taken (weight, height both standing and sitting, diameters, circumferences and skinfold thickness) from which different anthropometric indices were calculated (body mass index, Scelic and Grant indices, arm muscle circumference and area), and the somatotype was then determined. The body composition of each subject was assessed using the skinfold technique and the Jackson-Pollock (J-P) and Sloan-Weir (S-W) equations. The two groups of athletes showed very similar measurements regarding anthropometric characteristics. Only the Scelix index presented a significantly different value in the two groups (49.6+/-1.3 for group 1 vs. 51.1+/-1.3 for group 2; p<0.01). Group 1 showed a mesomorphic-ectomorphic somatotype, while the amateur athletes presented a balanced mesomorphic type. Moreover, a lower percentage of fat mass was more frequent in the first group (J-P=8.1+/-2.4%; S-W=8.9+/-3.3%) than in the second one (J-P=9.8+/-1.6%; S-W=11.2+/-3.7%), although the differences between the two groups were not significant. We conclude that group 1 is characterized by a slightly prominent vertical development of the skeletal frame. This could be an anthropometric characteristic that is best suited to meet the specific functional requirements of this sport. Moreover, both groups of athletes are characterized by a low percentage of fat mass, particularly the elite group.

Adult↗
PubMed2001

Copper trafficking: the solution structure of Bacillus subtilis CopZ.

A sequence with a high homology (39% residue identity) with that of the copper-transport CopZ protein from Enterococcus hirae and with the same MXCXXC metal-binding motif has been identified in the genome of Bacillus subtilis, and the corresponding protein has been expressed. The protein, constituted by 73 amino acids, does bind copper(I) under reducing conditions and fully folded in both copper-bound and copper-free forms under the present experimental conditions. The solution structure of the copper-bound form was determined through NMR spectroscopy on an 15N-labeled sample. A total of 1508 meaningful nuclear Overhauser effects, 38 dihedral phi angles, and 48 dihedral psi angles were used in the structural calculations, which lead to a family of 30 conformers with an average rmsd to the mean structure of 0.32 +/- 0.06 A for the backbone and of 0.85 +/- 0.07 A for the heavy atoms. NMR data on the apoprotein also show that, also in this form, the protein is in a folded state and essentially maintains the complete secondary structure. Some disorder is observed in the loop devoted to copper binding. These results are compared with those reported for CopZ from E. hirae whose structure is well-defined only in the apo form. The different behaviors of copper-loaded E. hirae and B. subtilis are tentatively accounted for on the basis of the presence of dithiothreitol used in the latter case, which would stabilize the monomeric form. The comparison is extended to other similar proteins, with particular attention to the copper-binding loop. The nature and the location of conserved residues around the metal-binding site are discussed with respect to their relevance for the metal-binding process. Proposals for the role of CopZ are also presented.

Amino Acid Sequence↗
PubMed2001

(15)N backbone dynamics of ferricytochrome b(562): comparison with the reduced protein and the R98C variant.

The backbone dynamics of ferricytochrome b(562), a four-helix bundle protein from Escherichia coli, have been studied by NMR spectroscopy. The consequences of the introduction of a c-type thioether linkage between the heme and protein and the reduction to the ferrous cytochrome have also been analyzed. (15)N relaxation rates R(1) and R(2) and (1)H-(15)N NOEs were measured at proton Larmor frequencies of 500 and 600 MHz for the oxidized and reduced protein as well as for the oxidized R98C variant. In the latter protein, an "artificial" thioether covalent bond has been introduced between the heme group and the protein frame [Arnesano, F., Banci, L., Bertini, I., Ciofi-Baffoni, S., de Lumley Woodyear, T., Johnson, C. M., and Barker, P. D. (2000) Biochemistry 39, 1499-1514]. The (15)N relaxation data were analyzed with the ModelFree protocol, and the mobility parameters on the picosecond to nanosecond time scale were compared for the three species. The three forms are rather rigid as a whole, with average generalized order parameters values of 0.87 +/- 0.08 (oxidized cytochrome b(562)), 0.84 +/- 0.07 (reduced cytochrome b(562)), and 0.85 +/- 0.07 (oxidized R98C cytochrome b(562)), indicating similar mobility for each system. Lower order parameters (S(2)) are found for residues belonging to loops 1 and 2. Higher mobility, as indicated by lower order parameters, is found for heme binding helices alpha 1 and alpha 4 in the R98C variant with respect to the wild-type protein. The analysis requires a relatively long rotational correlation time (tau(m) = 9.6 ns) whose value is accounted for on the basis of the anisotropy of the molecular shape and the high phosphate concentration needed to ensure the occurrence of monomer species. A parallel study of motions in the millisecond to microsecond time scale has also been performed on oxidized wild-type and R98C cytochrome b(562). In a CPMG experiment, decay rates were analyzed in the presence of spin-echo pulse trains of variable spacing. The dynamic behavior on this time scale is similar to that observed on the sub-nanosecond time scale, showing an increased mobility in the residues connected to the heme ligands in the R98C variant. It appears that the increased protein stability of the variant, established previously, is not correlated with an increase in rigidity.

Cytochrome b Group↗
PubMed2001

Backbone dynamics of plastocyanin in both oxidation states. Solution structure of the reduced form and comparison with the oxidized state.

A model-free analysis based on (15)N R(1), (15)N R(2), and (15)N-(1)H nuclear Overhauser effects was performed on reduced (diamagnetic) and oxidized (paramagnetic) forms of plastocyanin from Synechocystis sp. PCC6803. The protein backbone is rigid, displaying a small degree of mobility in the sub-nanosecond time scale. The loops surrounding the copper ion, involved in physiological electron transfer, feature a higher extent of flexibility in the longer time scale in both redox states, as measured from D(2)O exchange of amide protons and from NH-H(2)O saturation transfer experiments. In contrast to the situation for other electron transfer proteins, no significant difference in the dynamic properties is found between the two redox forms. A solution structure was also determined for the reduced plastocyanin and compared with the solution structure of the oxidized form in order to assess possible structural changes related to the copper ion redox state. Within the attained resolution, the structure of the reduced plastocyanin is indistinguishable from that of the oxidized form, even though small chemical shift differences are observed. The present characterization provides information on both the structural and dynamic behavior of blue copper proteins in solution that is useful to understand further the role(s) of protein dynamics in electron transfer processes.

Amino Acid Sequence↗
PubMed2001

Characterization of the binding interface between the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase.

The interaction of the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase, Ccc2a, was investigated by NMR in solution. In particular, a solution of Cu(I)-15NAtx1 was titrated with apo-Ccc2a, and, vice versa, a solution of Cu(I)-15NCcc2a was titrated with apo-Atx1. By following the 15N and 1H chemical shifts, a new species is detected in both experiments. This species is the same in both titrations and is in fast exchange with the parent species on the NMR time scale. Nuclear relaxation data are consistent with the formation of an adduct. Judging from the nuclear Overhauser effect spectroscopy patterns, the structure of Cu(I)-15NCcc2a in the presence of apo-Atx1 is not significantly altered, whereas Cu(I)-15NAtx1 in the presence of apo-Ccc2a experiences some changes with respect to both the apoproteins and the Cu(I)-loaded proteins. The structure of the Cu(I)-15NAtx1 moiety in the adduct was obtained from 1137 nuclear Overhauser effects to a final root mean square deviation to the mean structure of 0.76 +/- 0.13 A for the backbone and 1.11 +/- 0.11 A for the heavy atoms. 15N and 1H chemical shifts suggest the regions of interaction that, together with independent information, allow a structural model of the adduct to be proposed. The apo form of Atx1 displays significant mobility in loops 1 and 5, the N-terminal part of helix alpha1, and the C-terminal part of helix alpha2 on the ms-micros time scale. These regions correspond to the metal binding site. Such mobility is largely reduced in the free Cu(I)-Atx1 and in the adduct with apo-Ccc2a. The analogous mobility of Ccc2a in both Cu(I) and apo forms is reduced with respect to Atx1. Such an adduct is relevant as a structural and kinetic model for copper transfer from Atx1 to Ccc2a in physiological conditions.

Adenosine Triphosphatases↗
PubMed2001

Locating the metal ion in calcium-binding proteins by using cerium(III) as a probe.

The detection and assignment of NMR spectroscopic signals of carbon atoms from carbonyl and carboxylate groups in the loop hosting the Ce(III) ion was performed for the cerium-substituted calcium-binding protein calbindin D9k. This provided a tool to characterize in solution the first coordination sphere of the metal ion. Due to the well-documented possibility of replacing calcium with metal ions of the Ln(III) series, this approach turns out to be extremely efficient for characterizing in solution the coordination of calcium ions in proteins, independently of the availability of X-ray crystal structures. The present approach completes the structural characterization of lanthanide-substituted calcium-binding proteins, for which the role of long-range constraints arising from hyperfine interaction and self-orientation has already been assessed.

Calbindins↗
PubMed2001

Solvent (1)H NMRD study of hexaaquochromium(III): inferences on hydration and electron relaxation.

The water proton nuclear magnetic relaxation dispersions (NMRD) of hexaaquochromium(III) in water and in water-glycerol solutions were obtained at several temperatures and viscosities. The data were analyzed in terms of the available theories by taking into account the contributions from first sphere, second sphere, and outer sphere water molecules. A meaningful analysis was possible by taking advantage of the structural model obtained from (17)O relaxation data, which was recently made available in the literature (Bleuzen, A.; Foglia, F.; Furet, E.; Helm, L.; Merbach, A.; Weber, J. J. Am. Chem. Soc. 1996, 118, 12 777). Dynamic parameters, like the molecular rotational time, the exchange time of the water protons of the first coordination sphere, the correlation time for electron relaxation, and the magnetic field dependence of electron relaxation were obtained. The possible contribution to water proton relaxivity of second sphere water molecules for some other hexaaqua complexes is also discussed.

Journal Article↗
PubMed2001

Magnetic susceptibility tensor anisotropies for a lanthanide ion series in a fixed protein matrix.

The full series of lanthanide ions (except the radioactive promethium and the S-state gadolinium) has been incorporated into the C-terminal calcium binding site of the dicalcium protein calbindin D(9k). A fairly constant coordination environment is maintained throughout the series. At variance with several lanthanide complexes with small chelating ligands investigated in the past, the large protein moiety provides a large number of NMR signals whose hyperfine shifts can be exclusively ascribed to pseudocontact shifts (PCS). The chemical shifts of 1H and 15N backbone and side chain amide NH groups were accurately measured through HSQC experiments. 1097 PCS were estimated from these by subtracting the diamagnetic contributions measured on HSQC spectra of either the 4f(0) lanthanum(III) or the 4f(14) lutetium(III) derivatives and used to define a quality factor for the structure. The differences in diamagnetic chemical shifts between the two diamagnetic blanks were relatively small, although some were not negligible especially for the nuclei closest to the metal center. These differences were used as a tolerance for the PCS. The magnetic susceptibility tensor anisotropies for each paramagnetic lanthanide ion were obtained as the result of the solution structure determination performed by using the NOEs of the cerium(III) derivative and the PCS of all lanthanides simultaneously. This set of reliable magnetic data permits an experimental assessment of Bleaney's theory relative to the magnetic properties for an extended series of lanthanide complexes in solution. All of the obtained tensors show some rhombicity, as could be expected from the lack of symmetry of the protein environment. The directions of the largest magnetic susceptibility component for Ce, Pr, Nd, Sm, Tb, Dy, and Ho and of the smallest magnetic susceptibility component for Eu, Er, Tm, and Yb were found to be all within 15 degrees from their average (within 20 degrees for Sm), confirming the essential similarity of the coordination environment for all lanthanides. Bleaney's theory is in excellent qualitative agreement with the observed pattern of axial anisotropies. Its quantitative agreement is substantially better than that suggested by previous analyses performed on more limited sets of PCS data for small lanthanide complexes, the so-called crystal field parameter varying only within +/-30% from one lanthanide to another. These variations are even smaller (+/-15%) if a reasonable T(-3) correction is taken into consideration. A knowledge of magnetic susceptibility anisotropy properties of lanthanides is essential in determining the self-orienting properties of lanthanide complexes in solution when immersed in magnetic fields.

Algorithms↗
PubMed2001

Adduct of acetylene at sulfur in an oxygen- and sulfur-bridged open cubane cluster complex of tungsten.

Formation of a single carbon-sulfur bond is described. The reaction of incomplete cubane-type sulfur and oxygen-bridged isothiocyanato tungsten cluster [W3(mu3-S)(mu-O)(mu-S)2(NCS)9]5- (7) with acetylene affords [W3(mu3-S)(mu-O)(mu-S)(mu-SCH=CH2)(NCS)9]4- (8). The cluster 8 has been isolated as K0.5(Hpy)3.5[W3(mu3-S)(mu-O)(mu-S)(mu-SCH=CH2)(NCS)9] (8'), whose structure has been characterized by X-ray crystallography, electronic spectra, and 1H and 13C NMR spectroscopy. Crystal data of 8': triclinic system, space group P1, a = 14.465(5) A, b = 17.353(3) A, c = 10.202(2) A, alpha = 90.98(1) degrees, beta = 108.59(2) degrees, gamma = 98.13(2) degrees, V = 2397.6(10) A(3), Z = 2, D(c) = 2.096 g cm(-3), Dm = 2.08 g cm(-3), R (Rw) = 3.6 (5.5)% for 8786 reflections (I > 1.50 sigma(I)). The carbon-carbon distance is 1.27(1) A and is almost equidistant between ethylene (1.339 A) and acetylene (1.203 A). The electronic spectrum of 8' in 1.0 M HCl containing 1.5 M KSCN has a characteristic broad peak in the near-infrared region [lambda(max), nm (epsilon, M(-1) cm(-1)): 840 (650), 575 (1450)]. (1)H NMR and HH correlation spectroscopy (COSY) of 8' in CD3CN support the results of the X-ray structural analysis. The (1)H NMR spectrum shows three signals at 2.42 (1H, dd), 4.84 (1H, d, J = 8.8 Hz), and 4.89 (1H, d, J = 16.2 Hz) ppm due to the mu-SCH=CH2 moiety of 8'. The correlation spectrum shows spin couplings of the signal at 2.42 ppm with the signals at 4.84 and 4.89 ppm. The mechanism of the formation of 8 is suggested to proceed through an intermediate with acetylene bridging two of the sulfur atoms.

Journal Article↗
PubMed2001

The first solution structure of a paramagnetic copper(II) protein: the case of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803.

The NMR solution structure of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803 is here reported. The protein contains paramagnetic copper(II), whose electronic relaxation times are quite unfavorable for NMR solution studies. The structure has been solved on the basis of 1041 meaningful NOESY cross-peaks, 18 1D NOEs, 26 T(1) values, 96 dihedral angle constraints, and 18 H-bonds. The detection of broad hyperfine-shifted signals and their full assignment allowed the identification of the copper(II) ligands and the determination of the Cu-S-C-H dihedral angle for the coordinated cysteine. The global root-mean-square deviation from the mean structure for the solution structure family is 0.72 +/- 0.14 and 1.16 +/- 0.17 A for backbone and heavy atoms, respectively. The structure is overall quite satisfactory and represents a breakthrough, in that it includes paramagnetic copper proteins among the metalloproteins for which solution structures can be afforded. The comparison with the available X-ray structure of a triple mutant is also performed.

Amino Acid Sequence↗
PubMed2001

Solution structure of the Cu(I) and apo forms of the yeast metallochaperone, Atx1.

The (1)H NMR solution structure of the Cu(I)-bound form of Atx1, a 73-amino acid metallochaperone protein from the yeast Saccharomyces cerevisiae, has been determined. Ninety percent of the (1)H and 95% of the (15)N resonances were assigned, and 1184 meaningful NOEs and 42 (3)J(HNH)(alpha) and 60 (1)J(HN) residual dipolar couplings provided a family of structures with rmsd values to the mean structure of 0.37 +/- 0.07 A for the backbone and 0.83 +/- 0.08 A for all heavy atoms. The structure is constituted by four antiparallel beta strands and two alpha helices in a betaalphabetabetaalphabeta fold. Following EXAFS data [Pufahl, R., Singer, C. P., Peariso, K. L., Lin, S.-J., Schmidt, P. J., Fahrni, C. J., Cizewski Culotta, V., Penner-Hahn, J. E., and O'Halloran, T. V. (1997) Science 278, 853-856], a copper ion can be placed between two sulfur atoms of Cys15 and Cys18. The structure of the reduced apo form has also been determined with similar resolution using 1252 meaningful NOEs (rmsd values for the family to the mean structure are 0.67 +/- 0.12 A for the backbone and 1.00 +/- 0.12 A for all heavy atoms). Comparison of the Cu(I) and apo conformations of the protein reveals that the Cu(I) binding cysteines move from a buried site in the bound metal form to a solvent-exposed conformation on the surface of the protein after copper release. Furthermore, copper release leads to a less helical character in the metal binding site. Comparison with the Hg(II)-Atx1 solid-state structure [Rosenzweig, A. C., Huffman, D. L., Hou, M. Y., Wernimont, A. K., Pufahl, R. A., and O'Halloran, T. V. (1999) Structure 7, 605-617] provides insights into the copper transfer mechanism, and a pivotal role for Lys65 in the metal capture and release process is proposed.

Amino Acid Sequence↗
PubMed2001

Solution structure calculations through self-orientation in a magnetic field of a cerium(III) substituted calcium-binding protein.

Within the frame of a research aimed at characterizing paramagnetic metal ions capable of inducing self-orientation of metalloproteins in solution, we have studied the complex of the 75-amino-acid calcium-binding protein calbindin D(9k) with one Ce(III) ion (CaCeCb). Backbone (15)N-(1)H (1)J values have been determined for CaCeCb at two different magnetic fields. The above values showed a distinct dependence on the magnetic field, which is caused by the partial orientation of the molecule in solution. The difference in the values at the two magnetic fields provides structural constraints, which have been used to refine the structure of CaCeCb. The refined structure showed an improvement in terms of the number of residues falling in favored regions of the Ramachandran plot. The comparison of the molecular magnetic susceptibility tensor, obtained from the (15)N-(1)H (1)J values, with the magnetic susceptibility tensor of the metal, obtained from pseudocontact shifts, showed that the orientation of the molecule in solution is mainly determined by the Ce(III) ion. This paper shows that Ce(III), like low-spin Fe(III) in hemoproteins, is sufficiently magnetically anisotropic to induce self-orientation to an extent which can be exploited for solution structure determination.

Algorithms↗
PubMed2001

Cross correlation between the dipole-dipole interaction and the Curie spin relaxation: the effect of anisotropic magnetic susceptibility.

Cross-correlated relaxation caused by the interference of nuclear dipole-dipole interaction and the Curie spin relaxation (DD-CSR cross relaxation) is generalized to treat the case of anisotropic magnetic susceptibility, including the important case where the latter originates from zero-field splitting. It is shown that the phenomenon of DD-CSR cross relaxation is absolutely general and to be expected under any electronic configuration. The results of the generalization are presented for a model system, and the consequences for paramagnetic metalloproteins are illustrated with an example of cerium(III)-substituted calbindin. The effects of the magnetic anisotropy are found to be substantial.

Anisotropy↗
PubMed2001

Comparison and characterization of the [Fe4S4]2+/3+ centre in the wild-type and C77S mutated HiPIPs from Chromatium vinosum monitored by Mössbauer, 57Fe ENDOR and EPR spectroscopies.

Mössbauer, 57Fe ENDOR, CW and pulsed EPR experiments were performed on the reduced and the oxidized high-potential iron proteins (HiPIPs) of the wild type (WT) and the C77S mutant from Chromatium vinosum. The EPR spectra of the oxidized WT and mutant show three species respectively having nearly the same g-values but strongly changed spectral contributions. Relaxation times were estimated for oxidized WT and mutant at T = 5 K with pulsed EPR. A-tensor components of both iron pairs were obtained by 57Fe ENDOR, proving a similar magnetic structure for the WT and the mutant. Electronic relaxation has to be taken into account at T = 5 K in native and mutated oxidized HiPIPs to achieve agreement between Mössbauer and 57Fe ENDOR spectroscopies. The Mössbauer spectroscopy shows that the oxidized cluster contains a pure ferric and a mixed-valence iron pair coupled antiparallel. While all cluster irons from reduced C. vinosum WT are indistinguishable in the Mössbauer spectrum, the reduced C77S mutant shows a non-equivalence between the serine-bound and the three cysteine-ligated iron ions. The Mössbauer parameters confirm a loss of the covalent character of the iron bond when S is replaced by O and indicate a shift of the cluster's electron cloud towards the serine. Mössbauer spectra of the oxidized mutant can be simulated with two models: model I introduces a single electronic isomer with the serine always ligated to a ferric iron. Model II assumes two equally populated electronic isomers with the serine ligated to a ferric iron and a mixed-valence iron, respectively. The latter model is in better agreement with EPR and NMR.

Bacterial Proteins↗