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P Tavares

Publications and source records attributed to P Tavares.

At least 55 records · Page 3Linked to original sources
PubMed1997

Conversion of desulforedoxin into a rubredoxin center.

Rubredoxin and desulforedoxin both contain an Fe(S-Cys)4 center. However, the spectroscopic properties of the center in desulforedoxin differ from rubredoxin. These differences arise from a distortion of the metal site hypothesized to result from adjacent cysteine residues in the primary sequence of desulforedoxin. Two desulforedoxin mutants were generated in which either a G or P-V were inserted between adjacent cysteines. Both mutants exhibited optical spectra with maxima at 278, 345, 380, 480, and 560 nm while the low temperature X-band EPR spectra indicated highspin Fe3+ ions with large rhombic distortions (E/D = 0.21-0.23). These spectroscopic properties are distinct from wild type desulforedoxin and virtually identical to rubredoxin.

Cysteine↗
PubMed1996

Sequential headful packaging and fate of the cleaved DNA ends in bacteriophage SPP1.

The virulent Bacillus subtilis bacteriophage SPP1 packages its DNA from a precursor concatemer by a headful mechanism. Following disruption of mature virions with chelating agents the chromosome end produced by the headful cut remains stably bound to the phage tail. Cleavage of this tail-chromosome complex with restriction endonucleases that recognize single asymmetric positions within the SPP1 genome yields several distinct classes of DNA molecules whose size reflects the packaging cycle they were generated from. A continuous decrease in the number of molecules within each class derived from successive encapsidation rounds indicates that there are several packaging series which end after each headful packaging cycle. The frequency of molecules in each packaging class follows the distribution expected for a sequential mechanism initiated unidirectionally at a defined position in the genome (pac). The heterogeneity of the DNA fragment sizes within each class reveals an imprecision in headful cleavage of approximately 2.5 kb (5.6% of the genome size). The number of encapsidation events in a packaging series (processivity) was observed to increase with time during the infection process. DNA ejection through the tail can be induced in vitro by a variety of mild denaturing conditions. The first DNA extremity to exit the virion is invariably the same that was observed to be bound to the tail, implying that the viral chromosome is ejected with a specific polarity to penetrate the host. In mature virions a short segment of this chromosome end (55 to 67 bp equivalent to 187 to 288 A) is fixed to the tail area proximal to the head (connector). Upon ejection this extremity is the first to move along the tail tube to exit from the virion through the region where the tail spike was attached.

Bacillus Phages↗
PubMed1996

The triglyceride lowering effect of fish oils is affected by fish consumption.

We investigated the efficacy of fish oils in Portuguese patients with hypertriglyceridaemia and mixed hyperlipidaemia, and the influence of fish consumption on the triglyceride lowering capacity of fish oils. Forty patients participated in this double-blind study, consisting of a 4-week dietary or wash-out baseline period after which patients were randomly assigned to receive either 12 fish oil capsules (3.6 g/day of omega 3) or similar 12 soya oil capsules per day for a period of 2 months. There were no statistically significant changes of total, HDL or LDL-cholesterol, and triglycerides. Nevertheless, triglycerides increased 19.9% with soya oil and decreased 27.8% with fish oils. Also, there was an inverse relationship (rho = -0.352) between fish consumption and fish oils effect on triglycerides, and the triglyceride lowering with fish oils increased (from 27.8% to 44.4%), reaching borderline significance, if we excluded patients consuming one or more meals with fish per day. Glucose increased 11% (P = 0.0047) with fish oils. These findings suggest that the triglyceride lowering effect of fish oils is affected by fish consumption, and confirm that fish oils increase blood glucose levels in diabetics and non-diabetics.

Adolescent↗
PubMed1996

Intrinsic versus imposed curvature in cyclical oligomers: the portal protein of bacteriophage SPP1.

Large cyclical oligomers may be formed by (curvi-) linear polymerization of monomers until the n(th) monomer locks in with the first member of the chain. The subunits in incomplete structures exhibit a natural curvature with respect to each other which can be perturbed when the oligomer closes cyclically. Using cryo-electron microscopy and multivariate statistical image processing we report herein a direct structural observation of this effect. A sub-population (approximately 15%) of incomplete oligomers was found within a sample of SPP1 bacteriophage portal proteins embedded in vitreous ice. Whereas the curvature between adjacent subunits of the closed circular 13-fold symmetric oligomer is 27.7 degrees, in these incomplete oligomers the angle is only 25.8 degrees, a value which almost allows for a 14-subunit cyclical arrangement. A simple model for the association of large cyclical oligomers is suggested by our data.

Microscopy, Electron↗
PubMed1996

Primary structure of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774, a new class of non-heme iron proteins.

The primary structure of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774, a redox protein with two mononuclear iron sites, was determined by automatic Edman degradation and mass spectrometry of the composing peptides. It contains 125 amino acid residues of which five are cysteines. The first four, Cys-9, Cys-12, Cys-28 and Cys-29, are responsible for the binding of Center I which has a distorted tetrahedral sulfur coordination similar to that found in desulforedoxin from D. gigas. The remaining Cys-115 is proposed to be involved in the coordination of Center II, which is probably octahedrally coordinated with predominantly nitrogen/oxygen containing ligands as previously suggested by Mössbauer and Raman spectroscopy.

Amino Acid Sequence↗
PubMed1996

Preliminary crystallographic analysis of the oxidized form of a two mono-nuclear iron centres protein from Desulfovibrio desulfuricans ATCC 27774.

Crystals of the fully oxidized form of desulfoferrodoxin were obtained by vapor diffusion from a solution containing 20% PEG 4000, 0.1 M HEPES buffer, pH 7.5, and 0.2 M CaCl2. Trigonal and/or rectangular prisms could be obtained, depending on the temperature used for the crystal growth. Trigonal prisms belong to the rhombohedral space group R32, with a = 112.5 A and c = 63.2 A; rectangular prisms belong to the monoclinic space group C2, with a = 77.7 A, b = 80.9 A, c = 53.9 A, and beta = 98.1 degrees. The crystallographic asymmetric unit of the rhombohedral crystal form contains one molecule. There are two molecules in the asymmetric unit of the monoclinic form, in agreement with the self-rotation function.

Crystallization↗
PubMed1995

Crystal structure of desulforedoxin from Desulfovibrio gigas determined at 1.8 A resolution: a novel non-heme iron protein structure.

The crystal structure of desulforedoxin from Desulfovibrio gigas, a new homo-dimeric (2 x 36 amino acids) non-heme iron protein, has been solved by the SIRAS method using the indium-substituted protein as the single derivative. The structure was refined to a crystallographic R-factor of 16.9% at 1.8 A resolution. Native desulforedoxin crystals were grown from either PEG 4K or lithium sulfate, with cell constants a = b = 42.18 A, c = 72.22 A (for crystals grown from PEG 4K), and they belong to space group P3(2)21. The indium-substituted protein crystallized isomorphously under the same conditions. The 2-fold symmetric dimer is firmly hydrogen bonded and folds as an incomplete beta-barrel with the two iron centers placed on opposite poles of the molecule. Each iron atom is coordinated to four cysteinyl residues in a distorted tetrahedral arrangement. Both iron atoms are 16 A apart but connected across the 2-fold axis by 14 covalent bonds along the polypeptide chain plus two hydrogen bonds. Desulforedoxin and rubredoxin share some structural features but show significant differences in terms of metal environment and water structure, which account for the known spectroscopic differences between rubredoxin and desulforedoxin.

Amino Acid Sequence↗
PubMed1995

Expression of Desulfovibrio gigas desulforedoxin in Escherichia coli. Purification and characterization of mixed metal isoforms.

The dsr gene from Desulfovibrio gigas encoding the nonheme iron protein desulforedoxin was cloned using the polymerase chain reaction, expressed in Escherichia coli, and purified to homogeneity. The physical and spectroscopic properties of the recombinant protein resemble those observed for the native protein isolated from D. gigas. These include an alpha 2 tertiary structure, the presence of bound iron, and absorbance maxima at 370 and 506 nm in the UV/visible spectrum due to ligand-to-iron charge transfer bands. Low temperature electron paramagnetic resonance studies confirm the presence of a high-spin ferric ion with g values of 7.7, 5.7, 4.1, and 1.8. Interestingly, E. coli produced two forms of desulforedoxin containing iron. One form was identified as a dimer with the metal-binding sites of both subunits occupied by iron while the second form contained equivalent amounts of iron and zinc and represents a dimer with one subunit occupied by iron and the second with zinc.

Amino Acid Sequence↗
PubMed1995

Total synthesis of a simple metalloprotein-desulforedoxin.

Desulforedoxin is a protein purified from cellular extracts of Desulfovibrio gigas. It is a small (7.9 kDa) dimeric protein that contains a distorted rubredoxin like center (one single iron coordinated by four cysteinyl residues). Due to the simplicity of the polypeptide chain and of the iron center, an attempt was made to chemically produce this protein. A 36 amino acid polypeptide chain was synthesized based on the known sequence of native Desulforedoxin. The iron center was then reconstituted and the biochemical and spectroscopic characteristics of this synthetic protein were investigated. The final product has an equal sequence to the protein purified from D. gigas. The synthetic and natural Dx are very similar, in terms redox potential and spectroscopic properties (UV-Visible, EPR, Mössbauer).

Cysteine↗
PubMed1994

Spectroscopic properties of desulfoferrodoxin from Desulfovibrio desulfuricans (ATCC 27774).

Desulfoferrodoxin, a non-heme iron protein, was purified previously from extracts of Desulfovibrio desulfuricans (ATCC 27774) (Moura, I., Tavares, P., Moura, J. J. G., Ravi, N., Huynh, B. H., Liu, M.-Y., and LeGall, J. (1990) J. Biol. Chem. 265, 21596-21602). The as-isolated protein displays a pink color (pink form) and contains two mononuclear iron sites in different oxidation states: a ferric site (center I) with a distorted tetrahedral sulfur coordination similar to that found in desulforedoxin from Desulfovibrio gigas and a ferrous site (center II) octahedrally coordinated with predominantly nitrogen/oxygen-containing ligands. A new form of desulfoferrodoxin which displays a gray color (gray form) has now been purified. Optical, electron paramagnetic resonance (EPR), and Mössbauer data of the gray desulfoferrodoxin indicate that both iron centers are in the high-spin ferric states. In addition to the EPR signals originating from center I at g = 7.7, 5.7, 4.1, and 1.8, the gray form of desulfoferrodoxin exhibits a signal at g = 4.3 and a shoulder at g = 9.6, indicating a high-spin ferric state with E/D approximately 1/3 for the oxidized center II. Redox titrations of the gray form of the protein monitored by optical spectroscopy indicate midpoint potentials of +4 +/- 10 and +240 +/- 10 mV for centers I and II, respectively. Mössbauer spectra of the gray form of the protein are consistent with the EPR finding that both centers are high-spin ferric and can be analyzed in terms of the EPR-determined spin Hamiltonian parameters. The Mössbauer parameters for both the ferric and ferrous forms of center II are indicative of a mononuclear high spin iron site with octahedral coordination and predominantly nitrogen/oxygen-containing ligands. Resonance Raman studies confirm the structural similarity of center I and the distorted tetrahedral FeS4 center in desulforedoxin and provide evidence for one or two cysteinyl-S ligands for center II. On the basis of the resonance Raman results, the 635 nm absorption band that is responsible for the gray color of the oxidized protein is assigned to a cysteinyl-S-->Fe(III) charge transfer transition localized on center II. The novel properties and possible function of center II are discussed in relation to those of mononuclear iron centers in other enzymes.

Desulfovibrio↗
PubMed1993

The portal protein of bacteriophage SPP1: a DNA pump with 13-fold symmetry.

Electron microscopy in combination with image processing is a powerful method for obtaining structural information on non-crystallized biological macromolecules at the 10-50 A resolution level. The processing of noisy microscopical images requires advanced data processing methodologies in which one must carefully avoid the introduction of any form of bias into the data set. Using a novel multivariate statistical approach to the analysis of symmetry, we studied the structure of the bacteriophage SPP1 portal protein oligomer. This portal structure, ubiquitous in icosahedral bacteriophages which package dsDNA, is located at the site of symmetry mismatch between a 5-fold vertex of the icosahedral shell and the 6-fold symmetric (helical) tail. From previous studies such 'head-to-tail connector' structures were generally accepted to be homododecamers assembled in a 12-fold symmetric ring around a central channel. Using a new analysis methodology we have found that the phage SPP1 portal structure exhibits 13-fold cyclical symmetry: a new point group organization for oligomeric proteins. A model for the DNA packaging mechanism by 13-fold symmetric portal protein assemblies is presented which attributes a coherent functional meaning to their unusual symmetry.

Biological Transport↗
PubMed1992

Identification of a gene in Bacillus subtilis bacteriophage SPP1 determining the amount of packaged DNA.

The virulent Bacillus subtilis bacteriophage SPP1 encapsidates its DNA by a headful mechanism. Analyzing phage missense mutants, which package less DNA than SPP1 wild-type but show no other affected properties, we have identified a gene whose product is involved in the sizing of phage DNA during maturation. Characterization of this gene and its product provides an experimental access to the poorly understood mechanism of DNA sizing in packaging. The gene (gene 6 or siz) was cloned and sequenced. An open reading frame (ORF) coding for a 57.3 kDa polypeptide was identified. All the single nucleotide substitutions present in different siz mutants affect the net charge of that protein. The gene was further characterized by assignment of several nonsense mutations (sus) to the ORF. Phages carrying the latter type of mutations could be complemented in trans when gene 6 is provided by a plasmid.

Amino Acid Sequence↗
PubMed1992

Direct spectroscopic evidence for the presence of a 6Fe cluster in an iron-sulfur protein isolated from Desulfovibrio desulfuricans (ATCC 27774)

A novel iron-sulfur protein was purified from the extract of Desulfovibrio desulfuricans (ATCC 27774) to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a monomer of 57 kDa molecular mass. It contains comparable amounts of iron and inorganic labile sulfur atoms and exhibits an optical spectrum typical of iron-sulfur proteins with maxima at 400, 305, and 280 nm. Mössbauer data of the as-isolated protein show two spectral components, a paramagnetic and a diamagnetic, of equal intensity. Detailed analysis of the paramagnetic component reveals six distinct antiferromagnetically coupled iron sites, providing direct spectroscopic evidence for the presence of a 6Fe cluster in this newly purified protein. One of the iron sites exhibits parameters (delta EQ = 2.67 +/- 0.03 mm/s and delta = 1.09 +/- 0.02 mm/s at 140 K) typical for high spin ferrous ion; the observed large isomer shift indicates an iron environment that is distinct from the tetrahedral sulfur coordination commonly observed for the iron atoms in iron-sulfur clusters and is consistent with a penta- or hexacoordination containing N and/or O ligands. The other five iron sites are most probably high spin ferric. Three of them show parameters characteristic for tetrahedral sulfur coordination. In correlation with the EPR spectrum of the as-purified protein which shows a resonance signal at g = 15.3 and a group of signals between g = 9.8 and 5.4, this 6Fe cluster is assigned to an unusual spin state of 9/2 with zero field splitting parameters D = -1.3 cm-1 and E/D = 0.062. Other EPR signals attributable to minor impurities are also observed at the g = 4.3 and 2.0 regions. The diamagnetic Mössbauer component represents a second iron cluster, which, upon reduction with dithionite, displays an intense S = 1/2 EPR signal with g values at 2.00, 1.83, and 1.31. In addition, an EPR signal of the S = 3/2 type is also observed for the dithionite-reduced protein.

Amino Acid Sequence↗
PubMed1992

Photosensitivity to piroxicam: absence of cross-reaction with tenoxicam.

We studied 2 groups of patients. One group of 10 patients had a photosensitive eruption to piroxicam. Another group of 24 patients had positive patch test reactions to thimerosal and thiosalicylic acid and had never taken piroxicam or tenoxicam. Patients were patch tested with thimerosal 0.1% pet., thiosalicylic acid 0.1% pet., salicylic acid 2.0% pet., piroxicam 1 and 5% pet. and tenoxicam 1 and 5% pet. Photopatch tests were also performed with piroxicam and tenoxicam. All 10 patients with photosensitivity to piroxicam had positive patch tests to thimerosal and thiosalicylic acid and 9 of them had positive photopatch tests to piroxicam. 20 out of 24 patients with positive patch tests to thiosalicylic acid also had positive photopatch tests to piroxicam. All the patients tested with salicyclic acid were negative. Out of the 29 patients with positive photopatch tests to piroxicam, none reacted to tenoxicam. In countries with a high incidence of contact sensitivity to thimerosal/thiosalicylic acid, the use of piroxicam should be avoided and replaced by tenoxicam, a drug without reported photosensitivity.

Adult↗
PubMed1992

Tiaprofenic acid-induced photohemolysis in vitro is inhibited by nimesulide.

The effect of nimesulide on red blood cell (RBC) lysis photosensitized by tiaprofenic acid was investigated. The tiaprofenic acid-induced photohemolysis rate was enhanced by exposure to oxygen but lysis was also observed under anaerobic conditions. Photohemolysis was decreased by reduced glutathione (GSH) and reduced even more by butylated hydroxyanisole (BHA); sodium azide, superoxide dismutase and mannitol did not show a significant effect. Nimesulide did not cause any RBC lysis and inhibited this action of tiaprofenic acid by 20-30%, depending on the concentration of nimesulide and the intensity of ultraviolet A light. The protective effect of GSH, but not of BHA, was increased by nimesulide. Our findings suggest that free radicals are generated in this in vitro model of phototoxicity and are involved in the photoaggression to the red blood cell membrane, this effect being partially inhibited by nimesulide.

Anaerobiosis↗
PubMed1990

Purification and characterization of desulfoferrodoxin. A novel protein from Desulfovibrio desulfuricans (ATCC 27774) and from Desulfovibrio vulgaris (strain Hildenborough) that contains a distorted rubredoxin center and a mononuclear ferrous center.

A new type of non-heme iron protein was purified to homogeneity from extracts of Desulfovibrio desulfuricans (ATCC 27774) and Desulfovibrio vulgaris (strain Hildenborough). This protein is a monomer of 16-kDa containing two iron atoms per molecule. The visible spectrum has maxima at 495, 368, and 279 nm and the EPR spectrum of the native form shows resonances at g = 7.7, 5.7, 4.1 and 1.8 characteristic of a high-spin ferric ion (S = 5/2) with E/D = 0.08. Mössbauer data indicates the presence of two types of iron: an FeS4 site very similar to that found in desulforedoxin from Desulfovibrio gigas and an octahedral coordinated high-spin ferrous site most probably with nitrogen/oxygen-containing ligands. Due to this rather unusual combination of active centers, this novel protein is named desulfoferrodoxin. Based on NH2-terminal amino acid sequence determined so far, the desulfoferrodoxin isolated from D. desulfuricans (ATCC 27774) appears to be a close analogue to a recently discovered gene product from D. vulgaris (Brumlik, M.J., and Voordouw, G. (1989) J. Bacteriol. 171, 49996-50004), which was suggested to be a rubredoxin oxidoreductase. However, reduced pyridine nucleotides failed to reduce the desulforedoxin-like center of this new protein.

Amino Acid Sequence↗