Biomedical subjects

C M Soares

Publications and source records attributed to C M Soares.

At least 37 records · Page 2Linked to original sources

PubMed1999

The caa3 terminal oxidase of the thermohalophilic bacterium Rhodothermus marinus: a HiPIP:oxygen oxidoreductase lacking the key glutamate of the D-channel.

The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains a novel complex III and a high potential iron-sulfur protein (HiPIP) as the main electron shuttle (Pereira et al., Biochemistry 38 (1999) 1268-1275 and 1276-1283). In this paper, one of the terminal oxidases expressed in this bacterium is extensively characterised. It is a caa3-type oxidase, isolated with four subunits (apparent molecular masses of 42, 19 and 15 kDa and a C-haem containing subunit of 35 kDa), which has haems of the A(s) type. This oxidase is capable of using TMPD and horse heart cytochrome c as substrates, but has a higher turnover with HiPIP, being the first example of a HiPIP:oxygen oxidoreductase. The oxidase has unusually low reduction potentials of 260 (haem C), 255 (haem A) and 180 mV (haem A3). Subunit I of R. marinus caa3 oxidase has an overall significant homology with the subunits I of the COX type oxidases, namely the metal binding sites and most residues considered to be functionally important for proton uptake and pumping (K- and D-channels). However, a major difference is present: the putative essential glutamate (E278 in Paraccocus denitrificans) of the D-channel is missing in the R. marinus oxidase. Homology modelling of the R. marinus oxidase shows that the phenol group of a tyrosine residue may occupy a similar spatial position as the glutamate carboxyl, in relation to the binuclear centre. Moreover, sequence comparisons reveal that several enzymes lacking that glutamate have a conserved substitution pattern in helix VI: -YSHPXV- instead of -XGHPEV-. These observations are discussed in terms of the mechanisms for proton uptake and it is suggested that, in these enzymes, tyrosine may play the role of the glutamate in the proton channel.

Amino Acid Sequence↗

PubMed1999

An iterative structure-assisted approach to sequence alignment and comparative modeling.

Correct alignment of the sequence of a target protein with those of homologues of known three-dimensional structure is a key step in comparative modeling. Usually an iterative approach that takes account of the local and overall structural features is required. We describe such an approach that exploits databases of structural alignments of homologous proteins (HOMSTRAD, http:/(/)www-cryst.bioc.cam.ac.uk/ approximately homstrad) and protein superfamilies (CAMPASS, http:/(/)www-cryst.bioc.cam.ac.uk/ approximately campass), in which structure-based alignments are analyzed and formatted with the program JOY (http:/(/)www-cryst.bioc.cam.ac.uk/ approximately joy) to reveal conserved local structural features. The databases facilitate the recognition of a family or superfamily, they assist in the selection of useful parent structures, they are helpful in alignment of the target sequences with the parent set, and are useful for deriving relationships that can be used in validating models. In the iterative approach, a model is constructed on the basis of the proposed sequence alignment and this is then reexpressed in the JOY format and realigned with the parent set. This is repeated until the model and sequence alignment is optimized. We examine the case for comparison and use of multiple structures of family members, rather than a single parent structure. We use the targets attempted by our group in CASP3 to assess the value of such procedures.

Algorithms↗

PubMed1999

Improvement of side-chain modeling in proteins with the self-consistent mean field theory method based on an analysis of the factors influencing prediction.

With the objective of improving side-chain conformation prediction, we have analyzed the influence of various factors on prediction by the Self-Consistent Mean Field Theory method, applied to a set of high resolution x-ray protein structure models. These factors may be classed as variations in the mean field optimization protocol, variations in the potential energy function, and variations in rotamer library completeness. We have developed an optimization protocol that consistently reached lower mean field conformational free energies than two other protocols. This protocol led to an important improvement in prediction. We observed a major improvement in prediction with two more detailed van der Waals parameter sets, which we found to be due mainly to the introduction of scaling of 1-4 interactions. In a comparison of two knowledge-based rotamer libraries of considerably different size, we observed an unexpected decrease in prediction with an increase in library completeness. However, when we introduced a torsion potential term in the potential energy function, we found an important increase in average prediction and in the prediction of almost all residue types with a more complete rotamer set. The two knowledge-based rotamer libraries now became equivalent in terms of average prediction. The results we obtained in an analysis of the effect of the introduction of an additional electrostatic term in the potential energy function were largely inconclusive. However, we found a small increase in average prediction for an electrostatic potential term with a fixed dielectric constant of 15. The combined effect of all the factors we analyzed in this study resulted in average prediction accuracies of 79.9% for X1, 68.1% for X1 + 2, and 1.590 A for global rms deviation (RMSD); the corresponding values for core residues were 88.2%, 78.6%, and 1.171 A. These values represent improvements in average prediction of 6.5% for X1, 9.1% for X1 + 2, and 0.163 A for global RMSD over the original conditions; the corresponding improvements in the core were 5.9%, 9.0%, and 0.180 A, respectively.

Amino Acids↗

PubMed1999

Comparative redox and pKa calculations on cytochrome c3 from several Desulfovibrio species using continuum electrostatic methods.

A comparative study of the pH-dependent redox mechanisms of several members of the cytochrome c3 family has been carried out. In a previous work, the molecular determinants of this dependency (the so-called redox-Bohr effect) were investigated for one species using continuum electrostatic methods to find groups with a titrating range and strength of interaction compatible with a mediating role in the redox-Bohr effect. Here we clarify these aspects in the light of new and improved pKa calculations, our findings supporting the hypothesis of propionate D from heme I being the main effector in the pH-dependent modulation of the cytochrome c3 redox potentials in all the c3 molecules studied here. However, the weaker (but significant) role of other titrating groups cannot be excluded, their importance and identity changing with the particular molecule under study. We also calculate the relative redox potentials of the four heme centers among the selected members of the c3 family, using a continuum electrostatic method that takes into account both solvation and interaction effects. Comparison of the calculated values with available data for the microscopic redox potentials was undertaken, the quality of the agreement being dependent upon the choice of the dielectric constant for the protein interior. We find that high dielectric constants give best correlations, while low values result in better magnitudes for the calculated potentials. The possibility that the crystallographic calcium ion in c3 from Desulfovibrio gigas may be present in the solution structure was tested, and found to be likely.

Calcium↗

PubMed1999

Effects of protein-protein interactions on electron transfer: docking and electron transfer calculations for complexes between flavodoxin and c-type cytochromes.

Theoretical studies of protein-protein association and electron transfer were performed on the binary systems formed by Desulfovibrio vulgaris Hildenborough (D. v. H.) flavodoxin and D. v. H. cytochrome c553 and by flavodoxin and horse heart cytochrome c. Initial structures for the complexes were obtained by rigid-body docking and were refined by MD to allow for molecular flexibility. The structures thus obtained were analysed in terms of their relative stability through the calculation of excess energies. Electrostatic, van der Waals and solvation energy terms showed all to have significant contributions to the stability of complexes. In the best association solutions found for both cytochromes, these bind to different zones of flavodoxin. The binding site of flavodoxin observed for cytochrome c is in accordance with earlier works [27]. The various association modes found were characterised in terms of electron transfer using the Pathways model. For complexes between flavodoxin and horse heart cytochrome c, some correlation was observed between electron tunnelling coupling factors and conformation energy; the best conformation found for electron transfer corresponded also to the best one in terms of energy. For complexes between flavodoxin and cytochrome c553 this was not the case and a lower correlation was observed between electron tunnelling coupling factors and excess energies. These results are in accordance with the differences in the experimental dependence of electron transfer rates with ionic strength observed between these two cases.

Cytochrome c Group↗

PubMed1999

Nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774:primary sequence determination, crystallographic refinement at 1.8 and modelling studies of its interaction with the tetrahaem cytochrome c3.

A monomeric nine-haem cytochrome c (9Hcc) with 292 amino acid residues was isolated from cells of the sulfate- and nitrate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 grown under both nitrate- and sulfate-respiring conditions. The nucleotide sequence encoding the 292 residues was determined, allowing the correction of about 10% of the previous primary structure, determined from 1.8 A electron density maps. The refinement at 1.8 A resolution of the structural model was completed, giving an R-value of 16.5%. The nine haem groups are arranged into two tetrahaem clusters, located at both ends of the molecule, with Fe-Fe distances and local protein fold very similar to tetrahaem cytochromes c3, and the extra haem is located asymmetrically between the two regions. The new primary sequence determination confirmed the 39% sequence homology found between this cytochrome and the C-terminal region (residues 229-514) of the high-molecular-weight cytochrome c (Hmc) from D. vulgaris Hildenborough, providing strong evidence of structural similarity between 9Hcc and the C-terminal region of Hmc. The interaction between 9Hcc and the tetrahaem cytochrome c3 from the same organism was studied by modelling methods, and the results suggest that a specific interaction is possible between haem 4 of tetrahaem cytochrome c3 and haem 1 or haem 2 of 9Hcc, in agreement with previous kinetic experiments which showed the catalytic effect of the tetrahaem cytochrome c3 upon the reduction of 9Hcc by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774. These studies suggest a role for 9Hcc as part of the assembly of redox proteins involved in recycling the molecular hydrogen released by the cell as a result of substrate oxidation.

Amino Acid Sequence↗

PubMed1999

Simulation of electron-proton coupling with a Monte Carlo method: application to cytochrome c3 using continuum electrostatics.

A new method is presented for simulating the simultaneous binding equilibrium of electrons and protons on protein molecules, which makes it possible to study the full equilibrium thermodynamics of redox and protonation processes, including electron-proton coupling. The simulations using this method reflect directly the pH and electrostatic potential of the environment, thus providing a much closer and realistic connection with experimental parameters than do usual methods. By ignoring the full binding equilibrium, calculations usually overlook the twofold effect that binding fluctuations have on the behavior of redox proteins: first, they affect the energy of the system by creating partially occupied sites; second, they affect its entropy by introducing an additional empty/occupied site disorder (here named occupational entropy). The proposed method is applied to cytochrome c3 of Desulfovibrio vulgaris Hildenborough to study its redox properties and electron-proton coupling (redox-Bohr effect), using a continuum electrostatic method based on the linear Poisson-Boltzmann equation. Unlike previous studies using other methods, the full reduction order of the four hemes at physiological pH is successfully predicted. The sites more strongly involved in the redox-Bohr effect are identified by analysis of their titration curves/surfaces and the shifts of their midpoint redox potentials and pKa values. Site-site couplings are analyzed using statistical correlations, a method much more realistic than the usual analysis based on direct interactions. The site found to be more strongly involved in the redox-Bohr effect is propionate D of heme I, in agreement with previous studies; other likely candidates are His67, the N-terminus, and propionate D of heme IV. Even though the present study is limited to equilibrium conditions, the possible role of binding fluctuations in the concerted transfer of protons and electrons under nonequilibrium conditions is also discussed. The occupational entropy contributions to midpoint redox potentials and pKa values are computed and shown to be significant.

Binding Sites↗

PubMed1999

Differential expression of an hsp70 gene during transition from the mycelial to the infective yeast form of the human pathogenic fungus Paracoccidioides brasiliensis.

We have isolated and characterized cDNA and genomic clones that encode a 70 kDa heat shock protein (Hsp70) from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis. The gene encodes a 649-amino-acid protein showing high identity with other members of the hsp70 gene family. The hsp70 gene is induced during both heat shock of yeast cells at 42 degrees C and the mycelial to yeast transition. A differential expression of this gene can be observed between mycelial and yeast forms, with a much higher level of expression in the yeast. We found two introns of 178 and 72 nucleotides in the P. brasiliensis hsp70 gene. Splicing of these introns is regulated during the heat shock process and possibly during infection. In order to analyse the differential accumulation of unspliced mRNA following cellular differentiation and/or heat shock, reverse transcriptase-polymerase chain reaction (RT-PCR) experiments were carried out. The temperature-induced mycelial to yeast transition results in the transient accumulation of unspliced hsp70 mRNA transcripts. Yeast cells, after adaptation at 36 degrees C, seem to be more proficient at splicing, at least with respect to hsp70 mRNA because, during a severe heat shock (42 degrees C), the unspliced form of this mRNA does not accumulate. The mycelial to yeast differentiation will have the adaptational effect of increasing the resistance of the organism to environmental stress, which may be necessary for parasite survival in the mammalian host.

Amino Acid Sequence↗

PubMed1999

Local water bridges and protein conformational stability.

Recent studies have pointed out the important role of local water structures in protein conformational stability. Here, we present an accurate and computationally effective way to estimate the free energy contribution of the simplest water structure motif--the water bridge. Based on the combination of empirical parameters for accessible protein surface area and the explicit consideration of all possible water bridges with the protein, we introduce an improved protein solvation model. We find that accounting for water bridge formation in our model is essential to understand the conformational behavior of polypeptides in water. The model formulation, in fact, does not depend on the polypeptide nature of the solute and is therefore applicable to other flexible biomolecules (i.e., DNAs, RNAs, polysaccharides, etc.).

Hydrogen Bonding↗

PubMed1999

Characterization and utilization of Candida rugosa lipase immobilized on controlled pore silica.

Candida rugosa lipase was immobilized by covalent binding on controlled pore silica (CPS) using glutaraldehyde as cross-linking agent under aqueous and nonaqueous conditions. The immobilized C. rugosa was more active when the coupling procedure was performed in the presence of a nonpolar solvent, hexane. Similar optima pH (7.5-8.0) was found for both free and immobilized lipase. The optimum temperature for the immobilized lipase was about 10 degrees C higher than that for the free lipase. The thermal stability of the CPS lipase was also greater than the original lipase preparation. Studies on the operational stability of CPS lipase revealed good potential for recycling under aqueous (olive-oil hydrolysis) and nonaqueous (butyl butyrate synthesis) conditions.

Journal Article↗

PubMed1999

Fusel oil as precursor for aroma generation by biotransformation using lipase: scientific note.

The feasibility of using mixtures of C4 and C5 chain-length aliphatic alcohols from fusel oil as the bulk organic media for lipase-mediated synthesis of laurate esters was assessed. Reaction mixtures consisted of lauric acid, lipase, solvent (if added), and appropriate amount of fusel oil (previously dehydrated with inorganic salts and molecular sieves). The influence of the reaction conditions such as substrate concentrations and temperature were investigated. Increased molar ratio of acyl donor to acyl acceptor allowed the esterification to proceed with no need for solvent addition.

Journal Article↗

PubMed1999

Identification, N-terminal region sequencing and similarity analysis of differentially expressed proteins in Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is the causal agent of paracoccidioidomycosis, which is a systemic mycosis in Latin America. This human pathogen is a dimorphic fungus existing as mycelium (26 degrees C) and in infected tissues as a yeast form (36 degrees C). The in vitro differentiation process is reversible and dependent on temperature shift. In the present study, the total proteins from both forms of P. brasiliensis (isolate Pb01) were analysed by two-dimensional electrophoresis. Differentially expressed proteins were identified. Two of these proteins, PbM46 (mycelium) and PbY20 (yeast), were submitted to automated protein sequencing of their N-terminal regions. The 15 amino acid residue sequence of PbM46, AITKIFALKVYDSSG, is similar to enolases from several sources, and specially those from Saccharomyces cerevisiae (80%) and Candida albicans (67%), when compared to the NR database at NCBI using the BLASTP program. The 34 amino acid residue sequence of PbY20, APKIAIVFYSLYGHIQKLAEAQKKGIEAAGGTAD, could probably represent an allergen protein since it is very similar (90%) to the minor allergen protein of Alternaria alternata and 82% similar to the allergen protein of Cladosporium herbarum. This comparative analysis of proteins from mycelium and yeast forms has allowed the identification and characterization of differentially expressed proteins, probably related to differential gene expression in P. brasiliensis.

Amino Acid Sequence↗

PubMed1999

Virulence of Paracoccidioides brasiliensis isolates can be correlated to groups defined by random amplified polymorphic DNA analysis.

Fifteen Paracoccidioides brasiliensis isolates were discriminated by the RAPD analysis into two groups with only 17% of genomic identity. The ability of P. brasiliensis isolates to invade tissues was studied in an experimental model using susceptible B10.A mice. The analysis was performed according to the severity of the lesions including the number and size of the granuloma, the number and dissemination of fungi to different organs. The isolates from two RAPD groups demonstrated a marked difference in their virulence patterns for B10.A mice. The isolates Pb S, 662, Bt and 166 (group I) elicited localized infection restricted to the liver showing compact epithelioid granuloma with few fungi in the early post-infection period (slightly virulent). On the other hand the isolates Pb 01 and 7455 (group II) elicited a disseminated infection with a mixed suppurative and looser granulomatous inflammation, showing extensive areas of necrosis and large numbers of viable fungal cells (highly virulent). These results are strong evidence for correlation between RAPD patterns and the virulence degree of P. brasiliensis.

Animals↗

PubMed1998

Molecular dynamics simulation of cytochrome c3: studying the reduction processes using free energy calculations.

The tetraheme cytochrome c3 from Desulfovibrio vulgaris Hildenborough is studied using molecular dynamics simulation studies in explicit solvent. The high heme content of the protein, which has its core almost entirely made up of c-type heme, presents specific problems in the simulation. Instability in the structure is observed in long simulations above 1 ns, something that does not occur in a monoheme cytochrome, suggesting problems in heme parametrization. Given these stability problems, a partially restrained model, which avoids destruction of the structure, was created with the objective of performing free energy calculations of heme reduction, studies that require long simulations. With this model, the free energy of reduction of each individual heme was calculated. A correction in the long-range electrostatic interactions of charge groups belonging to the redox centers had to be made in order to make the system physically meaningful. Correlation is obtained between the calculated free energies and the experimental data for three of four hemes. However, the relative scale of the calculated energies is different from the scale of the experimental free energies. Reasons for this are discussed. In addition to the free energy calculations, this model allows the study of conformational changes upon reduction. Even if the precise details of the structural changes that take place in this system upon individual heme reduction are probably out of the reach of this study, it appears that these structural changes are small, similarly to what is observed for other redox proteins. This does not mean that their effect is minor, and one example is the conformational change observed in propionate D from heme I when heme II becomes reduced. A motion of this kind could be the basis of the experimentally observed cooperativity effects between heme reduction, namely positive cooperativity.

Biophysical Phenomena↗

PubMed1997

In vitro amphotericin B effects on growth, viability and dimorphism of Paracoccidioides brasiliensis: reversal of the treatment.

The in vitro effects of amphotericin B deoxycholate suspension (fungizone) on Paracoccidioides brasiliensis growth, cell viability and transformation were investigated. We also analyzed the protein synthesis patterns of both cellular forms, yeast and mycelium in the presence of AmB. This drug, at 30 micrograms/ml, highly inhibited yeast growth, which could be recovered depending on treatment time, where the most effective reversion was observed after 6 hr of incubation. The yeast cell viability, that had been partially affected by the drug, could also be efficiently recovered after AmB was removed. The effect of AmB on the cellular dimorphism process showed a strong reduction in the mycelium to yeast transformation (80% inhibition compared to the control without the drug). On the other hand, the transformation from yeast to mycelium in the presence of AmB was 50% affected, relative to the control. In contrast to the growth and cell viability experiments, the reversion effects on dimorphism were partial when the drug was removed, even with only 6 hr treatment. The two-dimensional gels of 35S-labeled proteins revealed a strong reduction in the three species of 80, 71 and 56 kDa in yeast and mycelium when treated with AmB.

Amphotericin B↗

PubMed1995

Ab initio determination of the crystal structure of cytochrome c6 and comparison with plastocyanin.

BACKGROUND: Electron transfer between cytochrome f and photosystem I (PSI) can be accomplished by the heme-containing protein cytochrome c6 or by the copper-containing protein plastocyanin. Higher plants use plastocyanin as the only electron donor to PSI, whereas most green algae and cyanobacteria can use either, with similar kinetics, depending on the copper concentration in the culture medium. RESULTS: We report here the determination of the structure of cytochrome c6 from the green alga Monoraphidium braunii. Synchrotron X-ray data with an effective resolution of 1.2 A and the presence of one iron and three sulfur atoms enabled, possibly for the first time, the determination of an unknown protein structure by ab initio methods. Anisotropic refinement was accompanied by a decrease in the 'free' R value of over 7% the anisotropic motion is concentrated at the termini and between residues 38 and 53. The heme geometry is in very good agreement with a new set of heme distances derived from the structures of small molecules. This is probably the most precise structure of a heme protein to date. CONCLUSIONS: On the basis of this cytochrome c6 structure, we have calculated potential electron transfer pathways and made comparisons with similar analyses for plastocyanin. Electron transfer between the copper redox center of plastocyanin to PSI and from cytochrome f is believed to involve two sites on the protein. In contrast, cytochrome c6 may well use just one electron transfer site, close to the heme unit, in its corresponding reactions with the same two redox partners.

Chlorophyta↗

PubMed1995

L3 loop-mediated mechanisms of pore closing in porin: a molecular dynamics perturbation approach.

L3 loop-mediated mechanisms for pore closing in porin are investigated with molecular dynamics simulation, using an approach that can be related to the phenomenon of voltage gating. Voltage gating is seen as a perturbation of the electrostatic screening inside the porin pore where, by the influence of the potential gradient, water and counter-ion distribution can be slightly displaced from their equilibrium distribution. This is simulated by perturbing the screening electrostatics of ionizable groups inside the pore. Under these conditions, a localized conformational change takes place, involving 12 (Ile102-Ala113) out of the 44 residues of the loop. The pore is reduced to a sixth of its open state size. The conformational change can be achieved with a small perturbation and it is reversible once the perturbation is switched off (relaxation process). Other types of behaviour predominating at higher simulation temperatures are found for the loop, involving an extra conformational change in the Thr92-Asp96 loop segment. This conformational change completely closes the pore, but is not reversible under the simulation conditions. Both zones involved in the conformational changes contain or overlap the zones which were described previously, using other techniques, to be the most flexible zones of the loop.

Computer Graphics↗

PubMed1995

Characterization of Paracoccidioides brasiliensis isolates by random amplified polymorphic DNA analysis.

We initially used 25 different random primers in order to test their ability to generate random amplified polymorphic DNA fragments from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis. From the tested primers we chose five to distinguish between seven isolates of this microorganism. The DNA amplification patterns allowed clear differentiation of the seven isolates into two distinct groups with only 35% genomic identity. One of these groups contained two subgroups with 81% genetic similarity. The random amplified polymorphic DNA analysis method proved to be a good tool for analyzing and comparing different genomes of P. brasiliensis isolates.

Base Sequence↗