Biomedical subjects

A Donaire

Publications and source records attributed to A Donaire.

At least 19 recordsLinked to original sources

PubMed2006

Cortical laminar necrosis related to prolonged focal status epilepticus.

Cortical laminar necrosis (CLN) is radiologically defined as high intensity cortical lesions on T1 weighted MRI images following a gyral distribution. Histopathologically, CLN is characterised by pannecrosis of the cortex involving neurones, glial cells, and blood vessels. It has been reported to be associated with hypoxia, metabolic disturbances, drugs, and infections. We present two patients who developed CLN and permanent neurological deficits after prolonged and repeated focal status epilepticus. The possible mechanisms leading to CLN in these patients are discussed, together with the implications of prompt and aggressive treatment in similar cases.

Adult↗

PubMed2006

[The utility of interictal SPECT in temporal lobe epilepsy].

INTRODUCTION: Interictal brain single photon emission computed tomography (SPECT) is used in the presurgical evaluation of patients with complex partial epilepsy. The aim of the present study was to compare interictal SPECT, MRI and video-electroencephalography (EEG) for seizure focus localization in patients with temporal lobe epilepsy, one year after temporal lobectomy, in order to determine the utility of interictal brain SPECT. PATIENTS AND METHODS: Thirty four consecutive patients with refractory temporal epilepsy were evaluated with video-EEG, MRI and interictal SPECT for seizure focus localization before surgery. Seizure focus was confirmed with the clinical follow-up one year after temporal lobectomy in all patients. MRI and SPECT analysis was performed visually. RESULTS: 31/34 patients were seizure free one year after surgery and the remaining 3 patients remain with seizures occasionally. Video-EEG results coincided with postsurgical seizure focus localization in 31 (91%) patients. MRI localized seizure focus correctly in 30 (88%) patients and was normal in 3 cases. Interictal brain SPECT was normal in 10 patients and showed temporal hypoperfusion consistent with postsurgical seizure focus in 23 (68%) patients. In all patients with abnormalities in the interictal SPECT, seizure focus was identified with video-EEG or MRI. CONCLUSIONS: When MRI and video-EEG localize seizure focus in the same temporal lobe, interictal brain SPECT does not offer any additional information for surgical decision making.

Adolescent↗

PubMed2005

Complex motor behaviors in temporal lobe epilepsy.

Complex motor behaviors differing from typical automatisms were found in 12 of 502 patients with temporal lobe epilepsy. Movements involved proximal limb segments (6) or body axis (6) and were often preceded by auras and followed by automatisms. Seven of 12 patients are seizure free after surgery. The other 5 patients declined surgery.

Adolescent↗

PubMed2001

Electronic characterization of the oxidized state of the blue copper protein rusticyanin by 1H NMR: is the axial methionine the dominant influence for the high redox potential?

The oxidized state of rusticyanin, the blue copper protein with the highest redox potential in its class, has been investigated through (1)H nuclear magnetic resonance applied to its cobalt(II) derivative. The assignment of the protons belonging to the coordinated residues has been performed. Many other amino acids situated in the vicinity of the metal ion, including six hydrophobic residues (isoleucine140 and five phenylalanines) have also been identified. The orientation of the main axes of the magnetic susceptibility tensor for the cobalt(II)-rusticyanin as well as its axial, Deltachi(ax), and rhombic, Deltachi(rh), magnetic susceptibility anisotropy components have been determined. A comparison of the present results with those previously obtained for cobalt(II)azurin [Donaire, A., Salgado, J., Moratal, J. M. (1998) Biochemistry 37, 8659-8673] allows us to provide further insights into the reasons for the high redox potential of this protein. According to our results, the interaction between the metal ion and the thioether Sdelta of the axial methionine is not as influential as the strong destabilizing effect that the hydrophobic residues close to the metal ion undergo in the oxidized state.

Anisotropy↗

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↗

PubMed2000

[Recurrent familial brachial plexopathy as the only clinical expression of neuropathy with susceptibility to pressure].

We report a family with hereditary neuropathy with liability to pressure palsies (HNPP) and chromosome 17p11.2 deletion. This family exhibits a peculiar phenotype consisting in recurrent brachial plexopathy episodes. This phenotype has to be distinguished from hereditary neuralgic amyotrophy on clinical grounds. Although the incidence of brachial plexopathy on HNPP is relatively high it is unusual as the sole symptom of the disease. It is noteworthy that in the six published families with this peculiar phenotype most of the acute episodes became evident after sleep. A greater liability of the plexus and a greater vulnerability to mechanical factors during sleep hours are the suggested mechanisms to explain this rare clinical onset. Recurrent painless brachial plexopathy when associated to generalized conduction abnormalities should suggest a HNPP.

Adolescent↗

PubMed1998

Metal coordination of azurin in the unfolded state.

1H NMR data applied to the paramagnetic cobalt(II) derivative of azurin from Pseudomonas aeruginosa have made it possible to show that the metal ion is bound to the protein in the unfolded state. The relaxation data as well as the low magnetic anisotropy of the metal ion indicate that the cobalt ion is tetrahedral in the unfolded form. The cobalt ligands have been identified as the residues Gly45, His46, Cys112 and His117. Met121 is not coordinated in the unfolded state. In this state, the metal ion is not constrained to adopt a bipyramidal geometry, as imposed by the protein when it is folded. This is clear confirmation of the rack-induced bonding mechanism previously proposed for the metal ion in azurin.

Azurin↗

PubMed1998

Solution structure of the oxidized Fe7S8 ferredoxin from the thermophilic bacterium Bacillus schlegelii by 1H NMR spectroscopy.

The solution structure of the paramagnetic seven-iron ferredoxin from Bacillus schlegelii in its oxidized form has been determined by 1H NMR. The protein, which contains 77 amino acids, is thermostable. Seventy-two residues and 79% of all theoretically expected proton resonances have been assigned. The structure has been determined through torsion angle dynamics calculations with the program DYANA, using 966 meaningful NOEs (from a total of 1305), hydrogen bond constraints, and NMR derived dihedral angle constraints for the cluster ligating cysteines, and by using crystallographic information to build up the two clusters. Afterwards, restrained energy minimization and restrained molecular dynamics were applied to each conformer of the family. The final family of 20 structures has RMSD values from the mean structure of 0.68 A for the backbone atoms and of 1.16 A for all heavy atoms. The contributions to the thermal stability of the B. schlegelii ferredoxin are discussed by comparing the present structure to that of the less stable Azotobacter vinelandii ferredoxin I which is the only other available structure of a bacterial seven-iron ferredoxin. It is proposed that the hydrophobic interactions and the hydrogen bond network linking the N-terminus and the C-terminus together and a high number of salt bridges contribute to the stability.

Amino Acid Sequence↗

PubMed1998

Determination of the magnetic axes of cobalt(II) and nickel(II) azurins from 1H NMR data: influence of the metal and axial ligands on the origin of magnetic anisotropy in blue copper proteins.

The orientation and the axial, Deltachiax, and rhombic, Deltachirh, components of the magnetic susceptibility tensor anisotropy for the cobalt(II) and nickel(II) derivatives of azurin from Pseudomonas aeruginosa have been determined from 1H NMR data. For both derivatives, the axial geometry of the system determines the orientation of the chi-tensor, whose z-axis forms an angle of 18.6 and 20.1 degrees with the Cu-OGly45 axial bond in the cobalt(II) and nickel(II) derivatives, respectively. For protons close to this axis, large negative pseudocontact shifts are observed, while those close to the NNS plane of the equatorial ligands experience lower and positive pseudocontact shifts for the same distance. Dipolar shifts are larger in the cobalt derivative, not only because of the larger spin number but also due to its intrinsically higher anisotropy. The contact contribution to the hyperfine shifts for the coordinated residues has been evaluated and analyzed in terms of unpaired spin delocalization mechanisms and geometry considerations. The results are extended to other blue copper proteins whose cobalt derivatives have been studied by 1H NMR. The electronic structure and its implications in the redox properties of the native copper proteins are also commented.

Anisotropy↗

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

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

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

Amino Acid Sequence↗

PubMed1996

Molecular model of the solution structure for the paramagnetic four-iron ferredoxin from the hyperthermophilic archaeon Thermococcus litoralis.

A molecular model for the three-dimensional solution structure of the paramagnetic, four-iron ferredoxin (Fd) from the hyperthermophilic archaeon Thermococcus litoralis (Tl) has been constructed on the basis of the reported 1H NMR spectral parameters [Donaire, A. (1996) J. Biomol. NMR 7, 35-47]. The conventional use of long mixing time NOESY cross-peak intensity, backbone angles, and hydrogenbonding constraints for building the structure was augmented by short mixing time NOESY, steady-state NOE, paramagnetic relaxation constraints, and the angular dependence of the ligated Cys H beta contact shifts. Distance geometry was used to generate various initial structures, and these structures were refined with the simulated annealing protocol. The family of structures with inconsequential violations exhibited low RMS deviations for the backbone except for a few residues in the immediate cluster vicinity and traces out a secondary structure very similar to those of the structurally characterized single cubane cluster Fds. The ability to describe the cluster environment depended on the use of numerous paramagnetic relaxation constraints which resulted in even the cluster loop residues exhibiting well-defined orientations, with the exception of one residue (Ilel1) whose 1H signals have not been located. Comparison of the structure of Tl Fd to those of mesophilic ferredoxins reveals that Tl Fd possesses the same secondary structural elements, two beta-sheets, two helices, and four turns, with the exception that the beta-sheet involving the termini incorporates a third strand in Tl Fd. Several minor structural adjustments in Tl Fd relative to other Fds, in addition to the third strand for beta-sheet, include the incorporation of the termini into the beta-sheet, a likely salt bridge from the side chain of the third beta-strand to the N-terminus, and a more hydrophobic and compact interaction between the large beta-sheet and the long helix. It is likely that each of these modifications, among others not yet well-defined (i.e., surface salt bridges), contributes to the extraordinary thermostability of Tl Fd.

Amino Acid Sequence↗

PubMed1996

1H NMR investigation of the secondary structure, tertiary contacts and cluster environment of the four-iron ferredoxin from the hyperthermophilic archaeon Thermococcus litoralis.

The solution molecular structure of the four-iron ferredoxin (Fd) from the hyperthermophilic archaeon Thermococcus litoralis (Tl) has been investigated by 1H NMR spectroscopy. TOCSY and NOESY experiments in H2O, tailored to detect both weakly and strongly relaxed resonances, together with steady-state NOEs in both H2O and D2O, allowed the identification of 58 of the 59 residues, with one residue near the paramagnetic center undetected. It is shown that the contact shifted and strongly relaxed signals for all four cysteines ligated to the paramagnetic cluster can be assigned by standard backbone connectivities that do not require any assumptions about the tertiary structure. Secondary structural elements identified in Tl Fd are a three-stranded antiparallel beta-strand involving the termini of the protein, a double beta-strand (also antiparallel), two alpha-helices and four turns. The existence of a disulfide bridge between the nonligated cysteines is also proposed. Dipolar contacts observed in the NOESY maps and by steady-state NOEs between the ligated cysteines and the 'diamagnetic' protein matrix indicate that the overall folding pattern of Tl Fd is very similar to that of the 3Fe ferredoxin from the mesophilic bacterium Desulfovibrio gigas [Kissinger et al. (1991) J. Mol. Biol., 219, 693-723]. The influence of the paramagnetism of the cluster on the relaxation properties of the proton signals of nonligated residues near the cluster, as well as on the ligated cysteines, correlates well with the proximity to the cluster iron(s), as predicted from the crystal structures for homologous protons of other single-cluster ferredoxins. Finally, the potential role of the various identified structural factors in contributing to the hyperthermostability of this protein is discussed.

Amino Acid Sequence↗

PubMed1995

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

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

Amino Acid Sequence↗

PubMed1995

1H-NMR study of a cobalt-substituted blue copper protein: Pseudomonas aeruginosa Co(II)-azurin.

Substitution of copper by cobalt in blue copper proteins gives a paramagnetic metalloderivative suitable for paramagnetic NMR studies. A thorough analysis of the 1H-NMR spectrum of Pseudomonas aeruginosa Co(II)-azurin is presented here. All the observable contact-shifted signals as well as many other paramagnetic signals from protons placed up to about 1.0 nm around the metal center, including some residues belonging to functionally important parts of the protein like the hydrophobic patch and the His35 region, have been assigned. The results obtained permit the detection and study of structural variations like those originated by the His35 ionization, and allow us to draw a feasible picture of the metal coordination site. Contact-shifted signals correspond to the same five residues which are found in the coordination sphere of the native Cu(II)-azurin, i.e. His46, His117, Cys112, Met121 and Gly45. Among them, the histidine residues present a pattern of resonances typical for histidines coordinated to cobalt in other cobalt protein derivatives, and the cysteine signals clearly indicate a strong interaction with the paramagnetic Co(II) ion. In contrast, the Met121 signals indicate a weak but still existent contact interaction with the metal center. On the other hand, the very weak copper ligand, Gly45, appears here as clearly coordinated to cobalt. Results are consistent with a distorted tetrahedral metal site with the cobalt deviated from the N2S plane towards the Gly45 O axial position and weakly interacting with the Met121 sulfur.

Azurin↗

PubMed1994

Interaction of cobalt ions with carboxypeptidase A.

The interaction of cobalt(II) with native and cobalt(II)-substituted carboxypeptidase has been investigated, at pH 7.5, by electronic absorption and 1H NMR spectroscopies. The reaction of the cobalt(II) uptake by the metalloenzyme [MCPA] (M = Zn or Co) occurs very slowly and a bimetallic complex, [MCPA(Co)], is formed. On the basis of the 1H NOE experiments, the isotropically shifted proton resonances were assigned as belonging to a coordinated histidine residue. 1H NMR titrations of [ZnCPA(Co)] with zinc(II) show that the zinc ion does not compete with cobalt for binding to the noncatalytic site. The temperature dependence of the isotropic shifts, molar absorbance, and longitudinal relaxation time values are indicative of a five-coordinated geometry for the cobalt ion. The identification of the noncatalytic cobalt binding site is also discussed.

Animals↗

PubMed1993

Two-dimensional 1H NMR spectra of ferricytochrome c551 from Pseudomonas aeruginosa.

The full assignment of 1H NMR signals of heme proton resonances of ferricytochrome c551 from Pseudomonas aeruginosa has been performed by means of 2D NMR experiments. This technique allows the complete and unequivocal assignment of all heme resonances, including methylene resonances of the propionic groups, directly implicated in the pH dependence of the redox properties of cytochrome c551.

Bacterial Proteins↗

PubMed1992

pH-dependent properties of cobalt(II) carboxypeptidase A-inhibitor complexes.

1H NMR spectroscopy of the isotropically shifted signals in cobalt carboxypeptidase, CoCPD, permits a direct and selective detection of protons belonging to the residues liganded to the metal. The chemical shift of these protons in the free enzyme and enzyme-inhibitor complexes with changing pH monitors the state of ionization of the ligands directly and of other residues in the active center indirectly. The 1H NMR spectrum of CoCPD at pH 6 shows three well-resolved isotropically shifted signals in the downfield region at 62 (a), 52 (c), and 45 (d) ppm which have been assigned to the NH proton of His-69 and to the C-4 H's of His-69 and His-196, respectively. Titration of signal a with pH is characterized by a pKa of 8.8 which is identical to that seen in prior electronic absorption and kinetic studies. The fact that the signal reflecting the NH of His-69 is still observed at pH 10 and no major shifts occur for the signals reflecting the C-4 H's indicates the alkaline pKa in carboxypeptidase A catalysis, pKEH, cannot be ascribed to ionization of the histidyl NH of either His-69 or His-196. Binding of L-Phe shifts this pKa to 7.7 while not greatly perturbing the downfield 1H NMR signals that reflect the ligation shell of the cobalt coordination sphere. These results indicate the pKa of 8.8 in CoCPD and the pKa of 7.7 in the CoCPD.L-Phe adduct reflect ionization of the same group.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗