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Veronica Morea

Publications and source records attributed to Veronica Morea.

12 recordsLinked to original sources
PubMed2006

Duplication, divergence and formation of novel protein topologies.

The rearrangement or permutation of protein substructures is an important mode of divergence. Recent work explored one possible underlying mechanism called permutation-by-duplication, which produces special forms of motif rearrangements called circular permutations. Permutation-by-duplication, involving gene duplication, fusion and truncation, can produce fully functional intermediate proteins and thus represents a feasible mechanism of protein evolution. In spite of this, circular permutations are relatively rare and we discuss possible reasons for their existence.

Amino Acid Motifs↗
PubMed2005

Insights into the catalytic mechanism of glutathione S-transferase: the lesson from Schistosoma haematobium.

Glutathione S-transferases (GSTs) are involved in detoxification of xenobiotic compounds and in the biosynthesis of important metabolites. All GSTs activate glutathione (GSH) to GS(-); in many GSTs, this is accomplished by a Tyr at H-bonding distance from the sulfur of GSH. The high-resolution structure of GST from Schistosoma haematobium revealed that the catalytic Tyr occupies two alternative positions, one external, involving a pi-cation interaction with the conserved Arg21, and the other inside the GSH binding site. The interaction with Arg21 lowers the pK(a) of the catalytic Tyr10, as required for catalysis. Examination of several other GST structures revealed the presence of an external pocket that may accommodate the catalytic Tyr, and suggested that the change in conformation and acidic properties of the catalytic Tyr may be shared by other GSTs. Arginine and two other residues of the external pocket constitute a conserved structural motif, clearly identified by sequence comparison.

Animals↗
PubMed2005

A novel thermostable hemoglobin from the actinobacterium Thermobifida fusca.

The gene coding for a hemoglobin-like protein (Tf-trHb) has been identified in the thermophilic actinobacterium Thermobifida fusca and cloned in Escherichia coli for overexpression. The crystal structure of the ferric, acetate-bound derivative, was obtained at 2.48 A resolution. The three-dimensional structure of Tf-trHb is similar to structures reported for the truncated hemoglobins from Mycobacterium tuberculosis and Bacillus subtilis in its central domain. The complete lack of diffraction patterns relative to the N- and C-terminal segments indicates that these are unstructured polypeptides chains, consistent with their facile cleavage in solution. The absence of internal cavities and the presence of two water molecules between the bound acetate ion and the protein surface suggest that the mode of ligand entry is similar to that of typical hemoglobins. The protein is characterized by higher thermostability than the similar mesophilic truncated hemoglobin from B. subtilis, as demonstrated by far-UV CD melting experiments on the cyano-met derivatives. The ligand-binding properties of Tf-trHb, analyzed in stopped flow experiments, demonstrate that Tf-trHb is capable of efficient O2 binding and release between 55 and 60 degrees C, the optimal growth temperature for Thermobifida fusca.

Actinobacteria↗
PubMed2005

Aminoacylation and conformational properties of yeast mitochondrial tRNA mutants with respiratory deficiency.

We report the identification and characterization of eight yeast mitochondrial tRNA mutants, located in mitochondrial tRNA(Gln), tRNA(Arg2), tRNA(Ile), tRNA(His), and tRNA(Cys), the respiratory phenotypes of which exhibit various degrees of deficiency. The mutations consist in single-base substitutions, insertions, or deletions, and are distributed all over the tRNA sequence and structure. To identify the features responsible for the defective phenotypes, we analyzed the effect of the different mutations on the electrophoretic mobility and efficiency of acylation of the mutated tRNAs in comparison with the respective wild-type molecules. Five of the studied mutations determine both conformational changes and defective acylation, while two have neither or limited effect. However, variations in structure and acylation are not necessarily correlated; the remaining mutation affects the tRNA conformation, but not its acylation properties. Analysis of tRNA structures and of mitochondrial and cytoplasmic yeast tRNA sequences allowed us to propose explanations for the observed defects, which can be ascribed to either the loss of identity nucleotides or, more often, of specific secondary and/or tertiary interactions that are largely conserved in native mitochondrial and cytoplasmic tRNAs.

Aminoacylation↗
PubMed2004

The truncated oxygen-avid hemoglobin from Bacillus subtilis: X-ray structure and ligand binding properties.

The group II truncated hemoglobin from Bacillus subtilis has been cloned, expressed, purified, and characterized. B. subtilis truncated hemoglobin is a monomeric protein endowed with an unusually high oxygen affinity (in the nanomolar range) such that the apparent thermodynamic binding constant for O2 exceeds that for CO by 1 order of magnitude. The kinetic basis of the high oxygen affinity resides mainly in the very slow rate of ligand release. The extremely stable ferrous oxygenated adduct is resistant to oxidation, which can be achieved only with oxidant in large excess, e.g. ferricyanide in 50-fold molar excess. The three-dimensional crystal structure of the cyano-Met derivative was determined at 2.15 A resolution. Although the overall fold resembles that of other truncated hemoglobins, the distal heme pocket displays a unique array of hydrophilic side chains in the topological positions that dominate the steric interaction with iron-bound ligands. In fact, the Tyr-B10, Thr-E7, and Gln-E11 oxygens on one side of the heme pocket and the Trp-G8 indole NE1 nitrogen on the other form a novel pattern of the "ligand-inclusive hydrogen bond network" described for mycobacterial HbO. On the proximal side, the histidine residue is in an unstrained conformation, and the iron-His bond is unusually short (1.91 A).

Amino Acid Sequence↗
PubMed2003

Exploiting evolutionary relationships for predicting protein structures.

In the last few years there have been many developments in computational biology, particularly with regard to novel, imaginative exploitation of genomic data. Disappointingly, there has been a lack of progress in the methodology for prediction of protein structures. In the last several years, however, promising new methods have finally begun to emerge. These methods are increasing the power and scope of the methodology, but, most importantly, they are generating new areas of investigation that we believe will accelerate progress in the field. In this review we describe recent developments and highlight the implications of their success as well as areas where efforts should be focused.

Amino Acid Sequence↗
PubMed2003

Vascular endothelial growth factor receptor-1 is deposited in the extracellular matrix by endothelial cells and is a ligand for the alpha 5 beta 1 integrin.

Vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase receptor for several growth factors of the VEGF family. Endothelial cells express a membrane-spanning form of VEGFR-1 and secrete a soluble variant of the receptor comprising only the extracellular region. The role of this variant has not yet been completely defined. In this study, we report that the secreted VEGFR-1 is present within the extracellular matrix deposited by endothelial cells in culture, suggesting a possible involvement in endothelial cell adhesion and migration. In adhesion assays, VEGFR-1 extracellular region specifically promoted endothelial cell attachment. VEGFR-1-mediated cell adhesion was divalent cation-dependent, and inhibited by antibodies directed against the alpha 5 beta 1 integrin. Moreover, VEGFR-1 promoted endothelial cell migration, and this effect was inhibited by anti-alpha 5 beta 1 antibodies. Direct binding of VEGFR-1 to the alpha 5 beta 1 integrin was also detected. Finally, binding to VEGFR-1 initiated endothelial cell spreading. Altogether these results indicate that the soluble VEGFR-1 secreted by endothelial cells becomes a matrix-associated protein that is able to interact with the alpha 5 beta 1 integrin, suggesting a new role of VEGFR-1 in angiogenesis, in addition to growth factor binding.

Amino Acid Sequence↗
PubMed2003

Engineering stable cytoplasmic intrabodies with designed specificity.

Many attempts have been made to develop antibody fragments that can be expressed in the cytoplasm ("intrabodies") in a stable and functional form. The recombinant antibody fragment scFv(F8) is characterised by peculiarly high in vitro stability and functional folding in both prokaryotic and eukaryotic cytoplasm. To dissect the relative contribution of different scFv(F8) regions to cytoplasmic stability and specificity we designed and constructed five chimeric molecules (scFv-P1 to P5) in which several groups of residues important for antigen binding in the poorly stable anti-hen egg lysozyme (HEL) scFv(D1.3) were progressively grafted onto the scFv(F8) scaffold. All five chimeric scFvs were expressed in a soluble form in the periplasm and cytoplasm of Escherichia coli. All the periplasmic oxidised forms and the scFv(P3) extracted from the cytoplasm in reducing conditions had HEL binding affinities essentially identical (K(d)=15nM) to that of the cognate scFv(D1.3) fragment (K(d)=16nM). The successful grafting of the antigen binding properties of D1.3 onto the scFv(F8) opens the road to the exploitation of this molecule as a scaffold for the reshaping of intrabodies with desired specificities to be targeted to the cytoplasm.

Amino Acid Sequence↗
PubMed2003

Exploring the cytochrome c folding mechanism: cytochrome c552 from thermus thermophilus folds through an on-pathway intermediate.

Understanding the role of partially folded intermediate states in the folding mechanism of a protein is a crucial yet very difficult problem. We exploited a kinetic approach to demonstrate that a transient intermediate of a thermostable member of the widely studied cytochrome c family (cytochrome c552 from Thermus thermophilus) is indeed on-pathway. This is the first clear indication of an obligatory intermediate in the folding mechanism of a cytochrome c. The fluorescence properties of this intermediate demonstrate that the relative position of the heme and of the only tryptophan residue cannot correspond to their native orientation. Based on an analysis of the three-dimensional structure of cytochrome c552, we propose an interpretation of the data which explains the residual fluorescence of the intermediate and is consistent with the established role played by some conserved interhelical interactions in the folding of other members of this family. A limited set of topologically conserved contacts may guide the folding of evolutionary distant cytochromes c through the same partially structured state, which, however, can play different kinetic roles, acting either as an intermediate or a transition state.

Cytochrome c Group↗
PubMed2003

Assessment of homology-based predictions in CASP5.

This report describes the assessment of the homology-based predictions submitted to the fifth edition of the Critical Assessment of Methods for Protein Structure Prediction (CASP5) experiment. We assessed the ability of the methods to predict the overall fold, the portions of the structure that differ substantially between the target protein and its closest structural homologue and the conformation of the side-chains. We also compared the results with those obtained in previous editions of the experiment and derived some general conclusions about the state of the art of comparative modeling methods and their usefulness for experimentalists.

Computational Biology↗
PubMed2002

Sequence conservation in families whose members have little or no sequence similarity: the four-helical cytokines and cytochromes.

Proteins for which there are good structural, functional and genetic similarities that imply a common evolutionary origin, can have sequences whose similarities are low or undetectable by conventional sequence comparison procedures. Do these proteins have sequence conservation beyond the simple conservation of hydrophobic and hydrophilic character at specific sites and if they do what is its nature? To answer these questions we have analysed the structures and sequences of two superfamilies: the four-helical cytokines and cytochromes c'-b(562). Members of these superfamilies have sequence similarities that are either very low or not detectable. The cytokine superfamily has within it a long chain family and a short chain family. The sequences of known representative structures of the two families were aligned using structural information. From these alignments we identified the regions that conserve the same main-chain conformation: the common core (CC). For members of the same family, the CC comprises some 50% of the individual structures; for the combination of both families it is 30%. We added homologous sequences to the structural alignment. Analysis of the residues occurring at sites within the CCs showed that 30% have little or no conservation, whereas about 40% conserve the polar/neutral or hydrophobic/neutral character of their residues. The remaining 30% conserve hydrophobic residues with strong or medium limitations on their volume variations. Almost all of these residues are found at sites that form the "buried spine" of each helix (at sites i, i+3, i+7, i+10, etc., or i, i+4, i+7, i+11, etc.) and they pack together at the centre of each structure to give a pattern of residue-residue contacts that is almost absolutely conserved. These CC conserved hydrophobic residues form only 10-15% of all the residues in the individual structures.A similar analysis of the cytochromes c'-b(562), which bind haem and have a very different function to that of the cytokines, gave very similar results. Again some 30% of the CC residues have hydrophobic residues with strong or medium conservation. Most of these form the buried spine of each helix and play the same role as those in the cytokines. The others, and some spine residues bind the haem co-factor.

Automation↗