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I Bertini

Publications and source records attributed to I Bertini.

At least 37 records · Page 2Linked to original sources
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↗
PubMed2001

Dimethyl propionate ester heme-containing cytochrome b5: structure and stability.

A derivative of rat microsomal cytochrome b5, obtained by substitution of the native heme moiety with protoporphyrin IX dimethyl ester, has been characterized by 1H and 15N NMR spectroscopy. Besides the two usual A and B forms, which depend on the orientation of the heme in the prostethic group cavity, two other minor forms have been detected which presumably indicate different conformations of the vinyl side chains. The shifts of the heme methyls, as well as the directions of the rhombic axes of the magnetic susceptibility tensor, indicate a small difference in the orientation of the imidazole planes of the histidine axial ligands. The solution structure was determined by using 1,303 meaningful NOEs and 241 pseudocontact shifts, the latter being derived from the native reduced protein. A family of 40 energy-minimized conformers was obtained with average RMSD of 0.56+/-0.09 A and 1.04+/-0.12 A for backbone and heavy atoms, respectively, and distance and pseudocontact shift penalty functions of 0.50+/-0.07 A2 and 0.51+/-0.02 ppm2. The structure shows some changes around the cavity and in particular a movement of the 60-70 backbone segment owing to the absence of two hydrogen bonds between the Ser64 backbone NH and side-chain OH and the carboxylate oxygen of propionate-7, present in the native protein. The analysis of the NMR spectra in the presence of unfolding agents indicates that this protein is less stable than the native form. The decrease in stability may be the result of the loss of the two hydrogen bonds connecting propionate-7 to Ser64 in the native protein. The available data on the reduction potential and the electron transfer rates are discussed on the basis of the present structural data.

Amino Acid Sequence↗
PubMed2001

Effects of extrinsic imidazole ligation on the molecular and electronic structure of cytochrome c.

Although imidazole ligand binding to cytochrome c is not directly related to its physiological function, it has the potential to provide valuable information on the molecular and electronic structure of the protein. The solution structure of the imidazole adduct of oxidized horse heart cytochrome c (Im-cyt c) has been determined through 2D NMR spectroscopy. The Im-cyt c, 8 mM in 1.2 M imidazole solution at pH 5.7 and 313 K, provided altogether 2,542 NOEs (1,901 meaningful NOEs) and 194 pseudocontact shifts. The 35 conformers of the family show the RMSD values to the average structure of 0.063+/-0.007 nm for the backbone and 0.107+/-0.007 nm for all heavy atoms, respectively. The characterization of Im-cyt c is discussed in detail both in terms of structure and electronic properties. The replacement of the axial ligand Met80 with the exogenous imidazole ligand induces significant conformation changes in both backbone and side chains of the residues located in the distal axial ligand regions. The imidazole ligand binds essentially parallel to the imidazole of the proximal histidine, the two planes forming an angle of 8+/-7 degrees. The electron delocalization on the heme moiety and the magnetic susceptibility tensor are consistent with these structural features.

Amino Acid Sequence↗
PubMed2001

Side chain mobility as monitored by CH-CH cross correlation: the example of cytochrome b5.

The mobility of betaCH2 moieties in oxidized and reduced cytochrome b5 was studied by analyzing the 13C relaxation of the J-split components, in terms of C-H dipole-C-H dipole cross correlation rates. A 2D 13C-1H experiment is proposed to measure these rates that provide the internal effective reorientation correlation time for each CH2 moiety. It is found that higher mobility is present in the alpha helices forming the heme pocket. On the contrary, the beta strands, which form the hydrophobic core of the molecule, have the lowest mobility. The general pattern is the same for the oxidized and reduced species, indicating that any oxidation-dependent property detected for backbone NH moieties does not affect the CH2 mobility.

Bacterial Proteins↗
PubMed2001

Paramagnetism-based versus classical constraints: an analysis of the solution structure of Ca Ln calbindin D9k.

The relative importance of paramagnetism-based constraints (i.e. pseudocontact shifts, residual dipolar couplings and nuclear relaxation enhancements) with respect to classical constraints in solution structure determinations of paramagnetic metalloproteins has been addressed. The protein selected for the study is a calcium binding protein, calbindin D9k, in which one of the two calcium ions is substituted with cerium(III). From 1823 NOEs, 191 dihedral angles, 15 hydrogen bonds, 769 pseudocontact shifts, 64 orientational constraints, 26 longitudinal relaxation rates, plus 969 pseudocontact shifts from other lanthanides, a final family with backbone r.m.s.d. from the average of 0.25 A was obtained. Then, several families of structures were generated either by removing subsets of paramagnetism-based constraints or by removing increasing numbers of NOEs. The results show the relative importance of the various paramagnetism-based constraints and their good complementarity with the diamagnetic ones. Although a resolved structure cannot be obtained with paramagnetism-based constraints only, it is shown that a reasonably well resolved backbone fold can be safely obtained by retaining as few as 29 randomly chosen long-range NOEs using the standard version of the program PSEUDYANA.

Amino Acid Substitution↗
PubMed2001

A further clue to understanding the mobility of mitochondrial yeast cytochrome c: a (15)N T1rho investigation of the oxidized and reduced species.

A new approach was developed to overproduce 15N-enriched yeast iso-1-cytochrome c in the periplasm of Escherichia coli in order to perform a study of the motions in the ms-micros time scale on the oxidized and reduced forms through rotating frame 15N relaxation rates and proton/deuterium exchange studies. It is confirmed that the reduced protein is rather rigid whereas the oxidized species is more flexible. The regions of the protein that display increased internal mobility upon oxidation are easily identified by the number of residues experiencing conformational equilibria and by their exchange rates. These data complement the information already available in the literature and provide a comprehensive picture of the mobility in the protein. In particular, oxidation mobilizes the loop containing Met80 and, through specific contacts, affects the mobility of helix 3 and possibly of helix 5, and of a section of protein connecting the heme propionates to helix 2. The relevance of internal motions to molecular recognition and to the early steps of the unfolding process of the oxidized species is also discussed. In agreement with the reported data, subnanosecond mobility is found to be less informative than the ms-micros with respect to redox dependent properties.

Cytochrome c Group↗
PubMed2001

Maternal dietary PUFAs intake and human milk content relationships during the first month of lactation.

Maternal dietary fatty acids (FFAs) intake and corresponding human milk composition relationships have been assessed throughout the first month of lactation in 34 lactating women consecutively enrolled. All mothers were on their habitual diet. Food records (95 items) were administered to the mothers, six-times during the first month of lactation (1 day after delivery, 4, 7, 14, 21, and 28 days after colostrum appearance) and referred to maternal dietary intake of the day before. Milk collected on day 1 was considered as colostrum, day 4 and 7 samples as transitional milk, and day 14, 21 and 28 samples as mature milk. Five gas chromatographic analyses were performed on each sample. Statistics were made using Friedman's and Pearson's test. Maternal dietary saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) were significantly related to the corresponding milk pattern in the phase of transitional milk (P<0.01), while total polyunsaturated (PUFAs) content was significantly related only to the mature milk (P<0.01); in this phase about 42% of the variations occurring in PUFAs milk content can be related to variation of maternal PUFAs dietary intake. The results in the present study provide evidence of the relationships between maternal diet and milk composition. The degree of correlation between maternal diet and PUFAs milk content increases throughout milk maturational process and reaches significance only in mature milk. This would imply that advancing lactation, milk PUFAs provision sources gradually shift from adipose tissue catabolism to maternal diet.

Adipose Tissue↗
PubMed2001

Validity and reliability of a new portable telemetric calorimeter designed to measure oxygen consumption and carbon dioxide production.

OBJECTIVE: The purpose of the present study was to evaluate the precision and the accuracy of a new portable telemetric calorimeter developed for the measurement of oxygen consumption, carbon dioxide production and pulmonary ventilation. DESIGN: An in vitro validation study. METHODS: An experimental protocol was designed to generate a series of tidal volumes (from 100 ml to 6000 ml at 200 ml steps) at different respiratory rates (5, 8, 14, 20, 40, 60, 75 breaths-min(-1)). For this purpose, a standardized pulmonary waveform generator system was utilized. Moreover, in order to evaluate the measure of O2 and CO2 concentrations at different temperatures (-10 degrees, 0 degrees, 15 degrees, 25 degrees, 40 degrees C), the instrument was placed inside an adjustable temperature and humidity chamber. RESULTS: The accuracy of flow measurements was within +/-3% in respect to the real values throughout the entire range of physiological values, whereas the measurements of gas concentrations were within +/-1% between 0 and 25 degrees C. CONCLUSIONS: The new portable telemetric calorimeter represents an easy to use instrument for the measure of energy expenditure during the activities of daily living.

Calorimetry, Indirect↗
PubMed2000

Solution structure of the yeast copper transporter domain Ccc2a in the apo and Cu(I)-loaded states.

Ccc2 is an intracellular copper transporter in Saccharomyces cerevisiae and is a physiological target of the copper chaperone Atx1. Here we describe the solution structure of the first N-terminal MTCXXC metal-binding domain, Ccc2a, both in the presence and absence of Cu(I). For Cu(I)-Ccc2a, 1944 meaningful nuclear Overhauser effects were used to obtain a family of 35 structures with root mean square deviation to the average structure of 0.36 +/- 0.06 A for the backbone and 0.79 +/- 0.05 A for the heavy atoms. For apo-Ccc2a, 1970 meaningful nuclear Overhauser effects have been used with 35 (3)J(HNHalpha) to obtain a family of 35 structures with root mean square deviation to the average structure of 0.38 +/- 0.06 A for the backbone and 0.82 +/- 0.07 A for the heavy atoms. The protein exhibits a betaalphabetabetaalphabeta, ferrodoxin-like fold similar to that of its target Atx1 and that of a human counterpart, the fourth metal-binding domain of the Menkes protein. The overall fold remains unchanged upon copper loading, but the copper-binding site itself becomes less disordered. The helical context of the copper-binding site, and the copper-induced conformational changes in Ccc2a differ from those in Atx1. Ccc2a presents a conserved acidic surface which complements the basic surface of Atx1 and a hydrophobic surface. These results open new mechanistic aspects of copper transporter domains with physiological copper donor and acceptor proteins.

Amino Acid Sequence↗
PubMed2000

Model-free analysis of a thermophilic Fe(7)S(8) protein compared with a mesophilic Fe(4)S(4) protein.

15N T(1), T(2) and (1)H-(15)N NOE were measured for the thermophilic Fe(7)S(8) protein from Bacillus schlegelii and for the Fe(4)S(4) HiPIP protein from Chromatium vinosum, which is a mesophilic protein. The investigation was performed at 276, 300, and 330 K at 11.7 T for the former, whereas only the 298 K data at 14.1 T for the latter were acquired. The data were analyzed with the model-free protocol after correcting the measured parameters for the effect of paramagnetism, because both proteins are paramagnetic. Both thermophilic and mesophilic proteins are quite rigid, with an average value of the generalized order parameter S2at room temperature of 0.92 and 0.94 for Fe(7)S(8) and Fe(4)S(4) proteins, respectively. The analyzed nitrogens for the Fe(7)S(8) protein showed a significant decrease in S2with increasing temperature, and at the highest temperature >70% of the residues had an internal correlation time. This research shows that subnanosecond rigidity is not related to thermostability and provides an estimate of the effect of increasing temperature on this time scale.

Bacillus↗
PubMed2000

Backbone dynamics of human Cu,Zn superoxide dismutase and of its monomeric F50E/G51E/E133Q mutant: the influence of dimerization on mobility and function.

The backbone assignment of reduced human dimeric Cu,Zn superoxide dismutase (SOD) was performed on a sample 100% enriched in (15)N, (13)C and 70% enriched in (2)H. (15)N T(1), T(2), and T(1)(rho) and (15)N-(1)H NOE assignment was performed at 600 MHz proton frequency on both wild-type SOD and the monomeric F50E/G51E/E133Q mutant. This allowed a comparison of the mobility in the subnanosecond and in the millisecond to microsecond time scales of the two systems. Both proteins are rather rigid, although some breathing of the beta sheets is detected in the wild type dimer. The monomer displays large mobility in the loops in the first part of the sequence, in loop IVa where point mutations have been introduced and at the C-terminus. The dimeric wild type is rigidified at loop IVa and at the C-terminus. Only loop VII shows a higher mobility in the dimer (besides some individual NH moieties). Conformational equilibria are displayed in the monomeric form around cysteines 57 and 146, thus explaining the disorder of arginine 143 which is the most important residue in guiding O(2)(-) toward the copper ion. The larger mobility in the wild type form with respect to the monomer in the picosecond to nanosecond time scale of helix alpha1 and loop VIIb, which provides the correct electrostatic driving force for O(2)(-) in the active channel, has been discussed in terms of favoring the activity of SOD.

Amino Acid Substitution↗
PubMed2000

Monitoring mobility in the early steps of unfolding: the case of oxidized cytochrome b(5) in the presence of 2 M guanidinium chloride.

A Model-Free analysis of the (15)N relaxation properties of oxidized cytochrome b(5), a heme-containing electron-transfer protein, has been performed in 2 M guanidinium chloride (GdmCl), i.e., just before the heme is released by the action of denaturant. This analysis provides information on the mobility in the nano- to picosecond time range. A parallel study on the motions in the milli- to microsecond time scale has also been performed by analyzing rotating-frame (15)N relaxation rates. The protein contains a 60:40 ratio of two conformers (A and B) differing for the rotation of the heme group around the alpha-gamma meso axis. The effect of denaturant has been followed for both species, and the mobility properties have been compared with the analogous information in the absence of denaturant. To complete the picture, we also performed (15)N relaxation measurements and the Model-Free analysis of the native B form, whereas data on the A form [Dangi, B., Sarma, S., Yan, C., Banville, D. L., Guiles, R. D. (1998) J. Phys. Chem. B 102, 8201-8208], as well as rotating-frame measurements for both native forms [Banci, L., Bertini, I., Cavazza, C., Felli, I. C., Koulougliotis, D. (1998) Biochemistry 37, 12320-12330; Arnesano, F., Banci, L., Bertini, I., Felli, I. C., Koulougliotis, D. (1999) Eur. J. Biochem. 260, 347-354], are already available in the literature. It is found that GdmCl tends to increase the internal mobility, although some residues are rigidified on both time scales. In the milli- to microsecond time scale, the tendency to increased mobility is reflected in a decrease in the tau(ex) values rather than in the number of residues experiencing conformational equilibria. In the nano- to picosecond time scale, the tendency to increased mobility is indicated by an overall decrease in the S(2) values. Color pictures are reported to visually show these effects. On the fast time scale, the B form is more mobile than the A form, reflecting the different stability with respect to unfolding. The increase in mobility upon addition of denaturant largely occurs around the heme pocket, which facilitates the release of the heme. The relevance of the internal motions with respect to the early steps of the unfolding process is also analyzed and discussed.

Animals↗
PubMed2000

The CrIIL reduction of [2Fe-2S] ferredoxins and site of attachment of CrIII using 1H NMR and site-directed mutagenesis.

The recently reported NMR solution structure of FeIIIFeIII parsley FdI has made possible 2D NOESY NMR studies to determine the point of attachment of CrIIIL in FeIIIFeIII...CrIIIL. The latter Cr-modified product was obtained by reduction of FeIIIFeIII parsley and spinach FdI forms with [Cr(15-aneN4) (H2O)2]2+ (15-aneN4 = 1,4,8,12-tetraazacyclopentadecane), referred to here as CrIIL, followed by air oxidation and chromatographic purification. From a comparison of NMR cross-peak intensities of native and Cr-modified proteins, two surface sites designated A and B, giving large paramagnetic CrIIIL broadening of a number of amino acid peaks, have been identified. The effects at site A (residues 19-22, 27, and 30) are greater than those at site B (residues 92-94 and 96), which is on the opposite side of the protein. From metal (ICP-AES) and electrospray ionization mass spectrometry (EIMS) analyses on the Cr-modified protein, attachment of a single CrIIIL only is confirmed for both parsley and spinach FdI and FdII proteins. Electrostatic interaction of the 3+ CrIIIL center covalently attached to one protein molecule (charge approximately -18) with a second (like) molecule provides an explanation for the involvement of two regions. Thus for 3-4 mM FeIIIFeIII...CrIIIL solutions used in NMR studies (CrIIIL attached at A), broadening effects due to electrostatic interactions at B on a second molecule are observed. Experiments with the Cys18Ala spinach FdI variant have confirmed that the previously suggested Cys-18 at site A is not the site of CrIIIL attachment. Line broadening at Val-22 of A gives the largest effect, and CrIIIL attachment at one or more adjacent (conserved) acidic residues in this region is indicated. The ability of CrIIL to bind in some (parsley and spinach) but not all cases (Anabaena variabilis) suggests that intramolecular H-bonding of acidic residues at A is relevant. The parsley and spinach FeIIFeIII...CrIIIL products undergo a second stage of reduction with the formation of FeIIFeII...CrIIIL. However, the spinach Glu92Ala (site B) variant undergoes only the first stage of reduction, and it appears that Glu-92 is required for the second stage of reduction to occur. A sample of CrIIIL-modified parsley FeIIIFeIII Fd is fully active as an electron carrier in the NADPH-cytochrome c reductase reaction catalyzed by ferredoxin-NADP+ reductase.

Amino Acid Sequence↗