PubMed Health⌕ Search

Biomedical subjects

Adriana Zagari

Publications and source records attributed to Adriana Zagari.

At least 19 recordsLinked to original sources
PubMed2006

The effect of the osmolyte trimethylamine N-oxide on the stability of the prion protein at low pH.

A study of the effect of trimethylamine N-oxide on the stability of two recombinant forms of the prion protein PrP, an ovine full-length and a human truncated form, is here reported. Both thermal denaturation and denaturation at room temperature were analyzed at pH values above and below the pKa of trimethylamine N-oxide, which is close to 4.7. Surprisingly, results showed that not only is trimethylamine N-oxide able to decrease PrP thermal stability at low pH but it also acts as a strong denaturant at room temperature. Likely, this destabilization is due to the capability of the cationic form of trimethylamine N-oxide to interact with the protein backbone as well as to weaken electrostatic interactions which are important for PrP fold. These results constitute the first experimental measurement of the effect of trimethylamine N-oxide on PrP stability and provide an unambiguous proof of the destabilizing effect of this osmolyte on PrP at low pH.

Animals↗
PubMed2006

Water molecules as structural determinants among prions of low sequence identity.

The nature of the factors leading to the conversion of the cellular prion protein (PrP(C)) into its amyloidogenic isoform (PrP(Sc)) is still matter of debate in the field of structural biology. The NMR structures of non-mammalian PrP(C) (non-mPrP) from frog, chicken and turtle [Calzolai, L., Lysek, D.A., Perez, D.R., Guntert, P. and Wuthrich, K. (2005) Prion protein NMR structures of chickens, turtles, and frogs. Proc. Natl. Acad. Sci. USA 102, 651-655] have provided some new and valuable information on the scaffolding elements that preserve the PrP(C) folding, despite their low sequence identity with the mammalian prions (mPrP). The present molecular dynamics study of non-mPrP(C) focuses on the hydration properties of these proteins in comparison with the mammalian ones. The data reveal new insights in the PrP hydration and focus on the implications for PrP(C) folding stability and its propensity for interactions. In addition, for the first time, a role in disfavoring the PrP(C) aggregation is suggested for a conserved beta-bulge which is stabilized by the local hydration.

Amino Acid Sequence↗
PubMed2006

Chemical denaturation of the elongation factor 1alpha isolated from the hyperthermophilic archaeon Sulfolobus solfataricus.

The stability against chemical denaturants of the elongation factor EF-1alpha (SsEF-1alpha), a protein isolated from the hyperthermophilic archaeon Sulfolobus solfataricus has been characterized in detail. Indeed, the atypical shape of the protein structure and the unusual living conditions of the host organism prompted us to analyze the effect of urea and guanidine hydrochloride (GuHCl) on the GDP complex of the enzyme (SsEF-1alpha x GDP) by fluorescence and circular dichroism. These studies were also extended to the nucleotide-free form of the protein (nfSsEF-1alpha). Interestingly, the experiments show that the denaturation curves of both SsEF-1alpha forms present a single inflection point, which is indicative of a cooperative unfolding process with no intermediate species. Moreover, the chemically induced unfolding process of both SsEF-1alpha x GDP and nfSsEF-1alpha is fully reversible. Both SsEF-1alpha forms exhibit remarkable stability against urea, but they do not display a strong resistance to the denaturing action of GuHCl. These findings suggest that electrostatic interactions significantly contribute to SsEF-1alpha stability.

Bacterial Proteins↗
PubMed2006

Limited tendency of alpha-helical residues to form disulfide bridges: a structural explanation.

Disulfide bridges have an enormous impact on the structure of a large number of proteins and polypeptides. Understanding the structural basis that regulates their formation may be important for the design of novel peptide-based molecules with a specific fold and stability. Here we report a statistical analysis of the relationships between secondary structure and disulfide bond formation, carried out using a large database of protein structures. Our analyses confirm the observation sporadically reported in previous investigations that cysteine residues located in alpha-helices display a limited tendency to form disulfide bridges. The very low occurrence of the disulfide bond in all alpha-chains compared to all beta-chains indicates that this property is also evident when proteins with different topologies are investigated. Taking advantage of the large database that endorsed the analysis on relatively rare motifs, we demonstrate that cysteine residues embedded in 3(10) helices present a good tendency to form disulfide bonds. This result is somewhat surprising since 3(10) helices are commonly assimilated into alpha-helices. A plausible structural explanation for the observed data has been derived combining analyses of disulfide bond sequence separation and of the length of the different secondary structure elements.

Cysteine↗
PubMed2006

Polyproline helices in protein structures: A statistical survey.

A statistical survey of polyproline II (PPII) helices extracted from protein crystal structures is here reported. The average hydrophobicity of these helices is intermediate between those displayed by beta-strands and coil regions and is similar to that of alpha-helices. PPII helices with amphipathic properties have been identified and classified. Amino acid propensities for PPII helices derived in this study differ significantly from those previously reported. They show a little albeit significant correlation with propensities for alpha-helices whereas they are fully non-correlated to propensities for beta-sheets. Finally, PPII propensities have been correlated with amino acid frequencies in structural proteins, such as collagen and extensins.

Algorithms↗
PubMed2005

Crystallization and preliminary X-ray crystallographic analysis of the Sulfolobus solfataricus nucleotide-exchange factor 1beta.

The nucleotide-exchange factor isolated from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-1beta) consists of 90 residues and differs from eukaryal EF-1betas. The protein has been successfully crystallized using either microbatch-under-oil or vapour-diffusion methods. Crystals of native SsEF-1beta diffract to 1.97 A resolution and belong to space group P2(1)2(1)2, with unit-cell parameters a = 106.46, b = 54.87, c = 44.03 A. Diffraction data have also been collected from a selenomethionine derivative of SsEF-1beta at 1.83 A resolution. Model building using the phases derived from the MAD experiment is in progress.

Cloning, Molecular↗
PubMed2005

Crystallization and preliminary X-ray crystallographic analysis of Sulfolobus solfataricus thioredoxin reductase.

A thermostable thioredoxin reductase isolated from Sulfolobus solfataricus (SsTrxR) has been successfully crystallized in the absence and in the presence of NADP. Two different crystal forms have been obtained. Crystals of the form that yields higher resolution data (1.8 A) belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 76.77, b = 120.68, c = 126.85 A. The structure of the enzyme has been solved by MAD methods using the anomalous signal from the Se atoms of selenomethionine-labelled SsTrxR.

Adenosine Diphosphate↗
PubMed2005

Lessons from crystals grown in the Advanced Protein Crystallisation Facility for conventional crystallisation applied to structural biology.

The crystallographic quality of protein crystals that were grown in microgravity has been compared to that of crystals that were grown in parallel on earth gravity under otherwise identical conditions. A goal of this comparison was to assess if a more accurate 3D-structure can be derived from crystallographic analysis of the former crystals. Therefore, the properties of crystals prepared with the Advanced Protein Crystallisation Facility (APCF) on earth and in orbit during the last decade were evaluated. A statistical analysis reveals that about half of the crystals produced under microgravity had a superior X-ray diffraction limit with respect of terrestrial controls. Eleven protein structures could be determined at previously unachieved resolutions using crystals obtained in the APCF. Microgravity induced features of the most relevant structures are reported. A second goal of this study was to identify the cause of the crystal quality enhancement useful for structure determination. No correlations between the effect of microgravity and other system-dependent parameters, such as isoelectric point or crystal solvent content, were found except the reduced convection during the crystallisation process. Thus, crystal growth under diffusive regime appears to be the key parameter explaining the beneficial effect of microgravity on crystal quality. The mimicry of these effects on earth in gels or in capillary tubes is discussed and the practical consequences for structural biology highlighted.

Animals↗
PubMed2005

Prion and water: tight and dynamical hydration sites have a key role in structural stability.

The propensity to form fibril in disease-related proteins is a widely studied phenomenon, but its correlation, if any, with structural characteristics of the associated proteins is not clearly understood. However, the observation has been made that some proteins that readily form amyloid have a significant number of backbone H bonds that are exposed to solvent molecules, suggesting that these regions have a propensity toward protein interaction and aggregation [Fernandez, A. & Scheraga, H. A. (2003) Proc. Natl. Acad. Sci. USA 100, 113-118]. High-resolution x-ray structures of the sheep and human C-terminal prion protein have provided a useful description of surface and partially buried waters. By molecular dynamics simulations, we investigated the structural role of these water molecules. The solvent dynamical behavior on the protein surface reveals significant features about the stability and the potential interactions of the prion protein. The protein presents regions of tightly bound conserved waters that are necessary to hold in place local elements of the fold, as well as regions where the local water is in fast exchange with bulk water. These results are evidenced by a map of the spatial distribution entropy of the solvent around the protein. The particular behavior of the solvent around these regions may be crucial in the folding stability and in terms of aggregation loci.

Amyloid↗
PubMed2005

Correlation between omega and psi dihedral angles in protein structures.

The planarity of the peptide group is one of the fundamental features of protein structure that is described in every chemistry and biochemistry textbook. By surveying a dataset of 163 atomic resolution protein structures we here identify the stereochemical conditions that favor significant deformations of peptide bond planarity. In particular, we demonstrate that the values of the omega dihedral angle are strictly correlated to the values of the adjacent psi angle. This trend is also observed in highly strained states such as those occurring in vicinal disulfide bridges. These findings provide direct evidence for the mutual influence of the geometrical parameters that describe the protein structure.

Databases, Protein↗
PubMed2004

Imino acids and collagen triple helix stability: characterization of collagen-like polypeptides containing Hyp-Hyp-Gly sequence repeats.

The analysis of factors contributing to the stability of proteins is a subject of intense debate. Particularly challenging is the study of structural proteins, since their function is their structure. Among these is collagen, the key structural component of bones, skin, cartilage, tendons, and other connecting tissues. It is well established that the collagen triple helix is characterized by the presence of hydroxyproline, whose content modulates triple helix thermal stability according to the requirement of the host organism. Because of the complexity and the fibrous nature of collagen, data on the stability and structure of this protein have been mainly obtained by the use of collagen-like polypeptides. On the basis of CD characterization of collagen-like polypeptides we here show that the presence of Hyp at the X position of repeating triplets Hyp-Hyp-Gly stabilizes the triple helix significantly. This extra-stabilization has been ascribed, by using molecular modeling, to the formation of a hydrogen bond between Hyp residues belonging to the X and the Y positions of adjacent chains. This communication also provides a comprehensive interpretation of the ensemble of available data on polypeptides containing proline derivatives.

Collagen↗
PubMed2004

The crystal structure of Sulfolobus solfataricus elongation factor 1alpha in complex with magnesium and GDP.

Recent studies have shown that elongation factors extracted from archaea/eukarya and from eubacteria exhibit different structural and functional properties. Along this line, it has been demonstrated that, in contrast to EF-Tu, Sulfolobus solfataricus EF-1alpha in complex with GDP (SsEF-1alpha.GDP) does not bind Mg(2+), when the ion is present in the crystallization medium at moderate concentration (5 mM). To further investigate the role that magnesium plays in the exchange process of EF-1alpha and to check the ability of SsEF-1alpha.GDP to bind the ion, we have determined the crystal structure of SsEF-1alpha.GDP in the presence of a nonphysiological concentration (100 mM) of Mg(2+). The analysis of the coordination of Mg(2+) unveils the structural bases for the marginal role played by the ion in the nucleotide exchange process. Furthermore, nucleotide exchange experiments carried out on a truncated form of SsEF-1alpha, consisting only of the nucleotide binding domain, demonstrate that the low affinity of SsEF-1alpha.GDP for Mg(2+) is due to the local architecture of the active site and does not depend on the presence of the other two domains. Finally, considering the available structures of EF-1alpha, a detailed mechanism for the nucleotide exchange process has been traced. Notably, this mechanism involves residues such as His14, Arg95, Gln131, and Glu134, which are strictly conserved in all archaea and eukarya EF-1alpha sequences hitherto reported.

Amino Acid Sequence↗
PubMed2004

Human aldolase A natural mutants: relationship between flexibility of the C-terminal region and enzyme function.

We have identified a new mutation in the FBP (fructose 1,6-bisphosphate) aldolase A gene in a child with suspected haemolytic anaemia associated with myopathic symptoms at birth and with a subsequent diagnosis of arthrogryposis multiplex congenita and pituitary ectopia. Sequence analysis of the whole gene, also performed on the patient's full-length cDNA, revealed only a Gly346-->Ser substitution in the heterozygous state. We expressed in a bacterial system the new aldolase A Gly346-->Ser mutant, and the Glu206-->Lys mutant identified by others, in a patient with an aldolase A deficit. Analysis of their functional profiles showed that the Gly346Ser mutant had the same Km as the wild-type enzyme, but a 4-fold lower kcat. The Glu206-->Lys mutant had a Km approx. 2-fold higher than that of both the Gly346-->Ser mutant and the wild-type enzyme, and a kcat value 40% less than the wild-type. The Gly346-->Ser and wild-type enzymes had the same Tm (melting temperature), which was approx. 6-7 degrees C higher than that of the Glu206-->Lys enzyme. An extensive molecular graphic analysis of the mutated enzymes, using human and rabbit aldolase A crystallographic structures, suggests that the Glu206-->Lys mutation destabilizes the aldolase A tetramer at the subunit interface, and highlights the fact that the glycine-to-serine substitution at position 346 limits the flexibility of the C-terminal region. These results also provide the first evidence that Gly346 is crucial for the correct conformation and function of aldolase A, because it governs the entry/release of the substrates into/from the enzyme cleft, and/or allows important C-terminal residues to approach the active site.

Amino Acid Substitution↗
PubMed2004

Population shift vs induced fit: the case of bovine seminal ribonuclease swapping dimer.

Bovine seminal ribonuclease (BS-RNase) is a unique member of the pancreatic-like ribonuclease superfamily. This enzyme exists as two conformational isomers with distinctive biological properties. The structure of the major isomer is characterized by the swapping of the N-terminal segment (MxM BS-RNase). In this article, the crystal structures of the ligand-free MxM BS-RNase and its complex with 2'-deoxycitidylyl(3',5')-2'-deoxyadenosine derived from isomorphous crystals have been refined. Interestingly, the comparison between this novel ligand-free form and the previously published sulfate-bound structure reveals significant differences. In particular, the ligand-free MxM BS-RNase is closer to the structure of MxM BS-RNase productive complexes than to the sulfate-bound form. These results reveal that MxM BS-RNase presents a remarkable flexibility, despite the structural constraints of the interchain disulfide bridges and the swapping of the N-terminal helices. These findings have important implications to the ligand binding mechanism of MxM BS-RNase. Indeed, a population shift rather than a substrate-induced conformational transition may occur in the MxM BS-RNase ligand binding process.

Animals↗
PubMed2004

Characterization of collagen-like heterotrimers: implications for triple-helix stability.

This article deals with the effects of proline hydroxylation on collagen triple-helix stability, an issue that is still under discussion. To investigate the structural determinants of triple-helix stabilization by hydroxyproline (Hyp), we here characterized spectroscopically triple-helix heterotrimers containing both chains of (Pro-Pro-Gly)10 and (Pro-Hyp-Gly)10. Results are discussed in relation to the various triple-helix stabilization mechanisms.

Amino Acid Sequence↗
PubMed2004

Six novel alleles identified in Italian hereditary fructose intolerance patients enlarge the mutation spectrum of the aldolase B gene.

Hereditary fructose intolerance (HFI) is a recessively inherited disorder of carbohydrate metabolism caused by impaired functioning of human liver aldolase (B isoform; ALDOB). To-date, 29 enzyme-impairing mutations have been identified in the aldolase B gene. Here we report six novel HFI single nucleotide changes identified by sequence analysis in the aldolase B gene. Three of these are missense mutations (g.6846T>C, g.10236G>T, g.10258T>C), one is a nonsense mutation (g.8187C>T) and two affect splicing sites (g.8180G>C and g.10196A>G). We have expressed in bacterial cells the recombinant proteins corresponding to the g.6846T>C (p.I74T), g.10236G>T (p.V222F), and g.10258T>C (p.L229P) natural mutants to study their effect on aldolase B function and structure. All the new variants were insoluble; molecular graphics data suggest this is due to impaired folding.

Adult↗
PubMed2003

Crystal structure of a ternary complex of the alcohol dehydrogenase from Sulfolobus solfataricus.

The crystal structure of a ternary complex of the alcohol dehydrogenase from the archaeon Sulfolobus solfataricus (SsADH) has been determined at 2.3 A. The asymmetric unit contains a dimer with a NADH and a 2-ethoxyethanol molecule bound to each subunit. The comparison with the apo structure of the enzyme reveals that this medium chain ADH undergoes a substantial conformational change in the apo-holo transition, accompanied by loop movements at the domain interface. The extent of domain closure is similar to that observed for the classical horse liver ADH, although some differences are found which can be related to the different oligomeric states of the enzymes. Compared to its apo form, the SsADH ternary complex shows a change in the ligation state of the active site zinc ion which is no longer bound to Glu69, providing additional evidence of the dynamic role played by the conserved glutamate residue in ADHs. In addition, the structure presented here allows the identification of the substrate site and hence of the residues that are important in the binding of both the substrate and the coenzyme.

Alcohol Dehydrogenase↗