Biomedical subjects

Andrea Bernini

Publications and source records attributed to Andrea Bernini.

At least 19 recordsLinked to original sources

PubMed2006

NMR studies of lysozyme surface accessibility by using different paramagnetic relaxation probes.

Paramagnetic probes, whose approach to proteins can be monitored by nuclear magnetic resonance (NMR) studies, have been found to be of primary relevance for investigating protein surfaces' accessibility. Here, a Gd(III) neutral complex which contains two metal ions, [Gd2(L7)(H2O)2], is suggested as a paramagnetic probe particularly suited for systematic NMR investigation of protein surface accessibility, due to an expected high relaxivity and to the lack of electric charge which could favor specific interactions. Hen egg white lysozyme has been used as a model system to verify the absence of preferential approaches of this paramagnetic probe to specific protein moieties by comparing paramagnetic perturbation profiles of 1H-13C HSQC signals obtained in the presence of TEMPOL and [Gd2(L7)(H2O)2]. From the similarity of the measured paramagnetic perturbation profiles induced by the two different probes, specific interactions of [Gd2(L7)(H2O)2] with the enzyme could be ruled out. The large size of the latter probe is suggested to be responsible for the strong paramagnetic perturbations observed for CalphaH groups which are located in convex surface-exposed regions. The combined use of the two probes reveals fine details of the dynamics controlling their approach toward the protein surface.

Electron Spin Resonance Spectroscopy↗

PubMed2006

NMR studies of BPTI aggregation by using paramagnetic relaxation reagents.

Paramagnetic probes, whose approach to proteins can be monitored by nuclear magnetic resonance (NMR) studies, have been found of primary relevance for investigating protein surfaces accessibility. Here, paramagnetic probes are also suggested for a systematic investigation on protein aggregation. Bovine pancreatic trypsin inhibitor (BPTI) was used as a model system for aggregation by analyzing its interaction with TEMPOL and Gd(III)DTPA-BMA. Some of the measured paramagnetic relaxation rates of BPTI protons exhibited a reverse dependence on protein concentration, which can be attributed to the formation of transient BPTI aggregates.

Animals↗

PubMed2006

Stable peptide inhibitors prevent binding of lethal and oedema factors to protective antigen and neutralize anthrax toxin in vivo.

The lethal and oedema toxins produced by Bacillus anthracis, the aetiological agent of anthrax, are made by association of protective antigen with lethal and oedema factors and play a major role in the pathogenesis of anthrax. In the present paper, we describe the production of peptide-based specific inhibitors in branched form which inhibit the interaction of protective antigen with lethal and oedema factors and neutralize anthrax toxins in vitro and in vivo. Anti-protective antigen peptides were selected from a phage library by competitive panning with lethal factor. Selected 12-mer peptides were synthesized in tetra-branched form and were systematically modified to obtain peptides with higher affinity and inhibitory efficiency.

Amino Acid Sequence↗

PubMed2006

Tertiary structure prediction of SARS coronavirus helicase.

SARS coronavirus, SCV, has been recently responsible of a sudden and widespread infection which caused almost 800 victims. The limited amount of SCV protein structural information is partially responsible of the lack of specific drugs against the virus. Coronavirus helicases are very conserved and peculiar proteins which have been proposed as suitable targets for antiviral drugs, such as bananins, which have been recently shown to inhibit the SCV helicase in vitro. Here, the quaternary structure of SCV helicase has been predicted, which will provide a solid foundation for the rational design of other antiviral helicase inhibitors.

Binding Sites↗

PubMed2005

Three-dimensional computation of atom depth in complex molecular structures.

MOTIVATION: For a complex molecular system the delineation of atom-atom contacts, exposed surface and binding sites represents a fundamental step to predict its interaction with solvent, ligands and other molecules. Recently, atom depth has been also considered as an additional structural descriptor to correlate protein structure with folding and functional properties. The distance between an atom and the nearest water molecule or the closest surface dot has been proposed as a measure of the atom depth, but, in both cases, the 3D character of depth is largely lost. In the present study, a new approach is proposed to calculate atom depths in a way that the molecular shape can be taken into account. RESULTS: An algorithm has been developed to calculate intersections between the molecular volume and spheres centered on the atoms whose depth has to be quantified. Many proteins with different size and shape have been chosen to compare the results obtained from distance-based and volume-based depth calculations. From the wealth of experimental data available for hen egg white lysozyme, H/D exchange rates and TEMPOL induced paramagnetic perturbations have been analyzed both in terms of depth indexes and of atom distances to the solvent accessible surface. The algorithm here proposed yields better correlations between experimental data and atom depth, particularly for those atoms which are located near to the protein surface. AVAILABILITY: Instructions to obtain source code and the executable program are available either from http://sienabiografix.com or http://sadic.sourceforge.net CONTACT: niccolai@unisi.it SUPPLEMENTARY INFORMATION: http://www.Sienabiogzefix.com/publication.

Algorithms↗

PubMed2005

Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human.

The genomic sequences of severe acute respiratory syndrome coronaviruses from human and palm civet of the 2003/2004 outbreak in the city of Guangzhou, China, were nearly identical. Phylogenetic analysis suggested an independent viral invasion from animal to human in this new episode. Combining all existing data but excluding singletons, we identified 202 single-nucleotide variations. Among them, 17 are polymorphic in palm civets only. The ratio of nonsynonymous/synonymous nucleotide substitution in palm civets collected 1 yr apart from different geographic locations is very high, suggesting a rapid evolving process of viral proteins in civet as well, much like their adaptation in the human host in the early 2002-2003 epidemic. Major genetic variations in some critical genes, particularly the Spike gene, seemed essential for the transition from animal-to-human transmission to human-to-human transmission, which eventually caused the first severe acute respiratory syndrome outbreak of 2002/2003.

Amino Acid Substitution↗

PubMed2005

cDNA cloning and expression of the phytoene synthase gene in sunflower.

A PCR approach was used to isolate a phytoene synthase (Psy) cDNA from sunflower (Helianthus annuus L.). A reconstructed full-length sequence (1598 bp) of the Psy cDNA was obtained; it contained a 1242 bp CDS, 172-nucleotides of 5'-untranslated region (UTR), and 170-nucleotides of 3'-UTR. The predicted protein (46.8kDa) displayed a sequence of 414 amino acid residues with a putative transit sequence for plastid targeting in the N-terminal region. Phylogenetic analysis demonstrated that the sunflower Psy (HaPsy) clustered with the marigold (Tagetes erecta) Psy gene, with which it showed an overall amino acid identity of 93.7%. Moreover, the HaPsy sequence relates closely with other Psy sequences of higher plants. HaPsy was highly expressed in cotyledons, and young and mature leaves. In contrast, HaPsy transcript levels were comparatively lower in the stem and almost absent in the roots. The HaPsy transcript levels were influenced by leaf expansion, which suggested that the expression of the HaPsy gene is regulated during the process of leaf development. The role of HaPsy in controlling carotenoid biosynthesis is demonstrated by the concurrent increase of HaPsy transcript levels with the light-dependent enhanced carotenoid production in green tissues of sunflower.

Alkyl and Aryl Transferases↗

PubMed2005

Synthetic peptides outside the spike protein heptad repeat regions as potent inhibitors of SARS-associated coronavirus.

A novel severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. We previously isolated and characterized SARS-CoV and SARS-CoV-like viruses from human and animals, respectively, suggesting that SARS could be transmitted from wild/farmed animals to humans. Comparison of the viral genomes indicated that sequence variation between animal and human isolates existed mainly in the spike (S) gene. We hypothesized that these variations may underlie a change of binding specificity of the S protein to the host cells, permitting viral transmission from animals to humans. Here we report that four 20-mer synthetic peptides (S protein fragments), designed to span these sequence variation hotspots, exhibited significant antiviral activities in a cell line. SARS-CoV infectivity was reduced over 10 000-fold through pre-incubation with two of these peptides, while it was completely inhibited in the presence of three peptides. Molecular modelling of the SARS-CoV peplomer suggests that three of these antiviral peptides map to the interfaces between the three monomers of the trimeric peplomer rather than the heptad repeat region from which short peptides are known to inhibit viral entry. Our results revealed novel regions in the spike protein that can be targeted to inhibit viral infection. The peptides identified in this study could be further developed into antiviral drugs.

Amino Acid Sequence↗

PubMed2004

Prediction of quaternary assembly of SARS coronavirus peplomer.

The tertiary structures of the S1 and S2 domains of the spike protein of the coronavirus which is responsible of the severe acute respiratory syndrome (SARS) have been recently predicted. Here a molecular assembly of SARS coronavirus peplomer which accounts for the available functional data is suggested. The interaction between S1 and S2 appears to be stabilised by a large hydrophobic network of aromatic side chains present in both domains. This feature results to be common to all coronaviruses, suggesting potential targeting for drugs preventing coronavirus-related infections.

Amino Acid Substitution↗

PubMed2004

Antioxidant activity of ferulic acid alkyl esters in a heterophasic system: a mechanistic insight.

The antioxidant activity of some esters of ferulic acid with the linear fatty alcohols C7, C8 (branched and linear), C9, C11, C12, C13, C15, C16, and C18 has been studied in homogeneous and heterogeneous phases. Whereas in homogeneous phase all of the alkyl ferulates possessed similar radical-scavenging abilities, in rat liver microsomes they showed striking differences, the more effective being C12 (7) (IC50 = 11.03 M), linear C8 (3) (IC50 = 12.40 microM), C13 (8) (IC50 = 18.60 microM), and C9 (5) (IC50 = 19.74 microM), followed by C7 (2), C15 (9), C11 (6), branched C8 (4), C16 (10), and C18 (11) (ferulic acid was the less active, IC50 = 243.84 microM). All of the molecules showed similar partition coefficients in an octanol-buffer system. Three-dimensional studies (NMR in solution, modeling in vacuo) indicate that this behavior might be due to a different anchorage of the molecules with the ester side chain to the microsomal phospholipid bilayer and to a consequent different orientation/positioning of the scavenging phenoxy group outside the membrane surface against the flux of oxy radicals.

Alkylation↗

PubMed2004

Interdomain tilt angle determines integrin-dependent function of the ninth and tenth FIII domains of human fibronectin.

Integrins are an important family of signaling receptors that mediate diverse cellular processes. The binding of the abundant extracellular matrix ligand fibronectin to integrins alpha(5)beta(1) and alpha(v)beta(3) is known to depend upon the Arg-Gly-Asp (RGD) motif on the tenth fibronectin FIII domain. The adjacent ninth FIII domain provides a synergistic effect on RGD-mediated integrin alpha(5)beta(1) binding and downstream function. The precise molecular basis of this synergy remains elusive. Here we have dissected further the function of FIII9 in integrin binding by analyzing the biological activity of the FIII9-10 interdomain interface variants and by determining their structural and dynamic properties in solution. We demonstrate that the contribution of FIII9 to both alpha(5)beta(1) and alpha(v)beta(3) binding and downstream function critically depends upon the interdomain tilt between the FIII9 and FIII10 domains. Our data suggest that modulation of integrin binding by FIII9 may arise in part from its steric properties that determine accessibility of the RGD motif. These findings have wider implications for mechanisms of integrin-ligand binding in the physiological context.

Amino Acid Motifs↗

PubMed2004

NMR and MD studies on the interaction between ligand peptides and alpha-bungarotoxin.

The interaction between alpha-bungarotoxin and linear synthetic peptides, mimotope of the nicotinic acetylcholine receptor binding site, has been characterised extensively by several methods and a wealth of functional, kinetic and structural data are available. Hence, this system represents a suitable model to explore in detail the dynamics of a peptide-protein interaction. Here, the solution structure of a new complex of the protein toxin with a tridecapeptide ligand exhibiting high affinity has been determined by NMR. As observed for three other previously reported mimotope-alpha-bungarotoxin complexes, also in this case correlations between biological activity and kinetic data are not fully consistent with a static discussion of structural data. Molecular dynamics simulations of the four mimotope-toxin complexes indicate that a relevant contribution to the complex stability is given by the extent of the residual flexibility that the protein maintains upon peptide binding. This feature, limiting the entropy loss caused by protein folding and binding, ought to be generally considered in a rational design of specific protein ligands.

Bungarotoxins↗

PubMed2004

NMR studies of the inclusion complex between beta-cyclodextrin and paroxetine.

A 1H and 13C NMR study on the inclusion complex of paroxetine with beta-cyclodextrin was carried out in order to define the stoichiometry of the association and its strength. Proton and carbon chemical shift measurements of paroxetine and beta-cyclodextrin were performed at several molar ratios and temperatures, allowing the determination of a 1:1 stoichiometry and an association constant value of the order of 2 x 10(3) for the paroxetine-beta-cyclodextrin complex. Overhauser effects in the rotating frame were also measured, and the experimental interproton distance constraints have been used for molecular model building of the complex. The obtained model indicates that the benzodioxolyl moiety of paroxetine is deeply inserted in the cavity of the cylindrical structure of beta-cyclodextrin, while the fluoro-phenyl ring lays above the wider rim.

Freezing↗

PubMed2003

Molecular modelling of S1 and S2 subunits of SARS coronavirus spike glycoprotein.

The S1 and S2 subunits of the spike glycoprotein of the coronavirus which is responsible for the severe acute respiratory syndrome (SARS) have been modelled, even though the corresponding amino acid sequences were not suitable for tertiary structure predictions with conventional homology and/or threading procedures. An indirect search for a protein structure to be used as a template for 3D modelling has been performed on the basis of the genomic organisation similarity generally exhibited by coronaviruses. The crystal structure of Clostridium botulinum neurotoxin B appeared to be structurally adaptable to human and canine coronavirus spike protein sequences and it was successfully used to model the two subunits of SARS coronavirus spike glycoprotein. The overall shape and the surface hydrophobicity of the two subunits in the obtained models suggest the localisation of the most relevant regions for their activity.

Amino Acid Sequence↗

PubMed2003

NMR studies of protein hydration and TEMPOL accessibility.

Understanding the mechanisms of the interaction between a protein surface and its outer molecular environment is of primary relevance for the rational design of new drugs and engineered proteins. Protein surface accessibility is emerging as a new dimension of Structural Biology, since NMR methods have been developed to follow how molecules, even those different from physiological ligands, preferentially approach specific regions of the protein surface. Hen egg-white lysozyme, a paradigmatic example of the state of the art of protein structure and dynamics, has been selected as a model system to study protein surface accessibility. Bound water and soluble spin-labels have been used to investigate the interaction of this enzyme, both free and bound to the inhibitor (NAG)(3), with its molecular environment. No tightly bound water molecules were found inside the enzyme active site, which, conversely, appeared as the most exposed to visits from the soluble paramagnetic probe TEMPOL. From the presented set of data, an integrated view of lysozyme surface accessibility towards water and TEMPOL molecules is obtained.

Binding Sites↗

PubMed2003

Biochemical filtering of a protein-protein docking simulation identifies the structure of a complex between a recombinant antibody fragment and alpha-bungarotoxin.

The structural characterization of a complex of alpha-bungarotoxin with a recombinant antibody fragment that mimics the acetylcholine receptor was achieved using docking simulation procedures. To drive the computer simulation towards a limited set of solutions with biological significance, a filter, incorporating general considerations of antigen-antibody interactions, specificity of the selected antibody fragment and results from alpha-bungarotoxin epitope mapping, was adopted. Two similar structures were obtained for the complex, both of them stabilized by cation-pi and hydrophobic interactions due to tyrosilyl residues of the antibody fragment. Site-directed mutagenesis studies, removing each of the latter aromatic residues and causing full inactivation of the interaction process between the antibody fragment and the neurotoxin, support the validity of the calculated structure of the complex.

Amino Acid Sequence↗

PubMed2003

Structure and function correlations between the rat liver threonine deaminase and aminotransferases.

The rat liver threonine deaminase is a cytoplasmic enzyme that catalyses the pyridoxal-phosphate-dependent dehydrative deamination of L-threonine and L-serine to ammonia and alpha-ketobutyrate and pyruvate, respectively, in vivo. During deamination, a molecule of the cofactor is converted to pyridoxamine phosphate. Recently, the ability of this enzyme to accomplish an inverse half-reaction, restoring pyridoxal-phosphate and L-alanine or L-aminobutyrate, respectively, from pyruvate or 2-oxobutyrate, was reported. In order to investigate the molecular mechanisms of this transaminating activity, a molecular model of rat liver threonine deaminase was constructed on the basis of sequence homology with the biosynthetic threonine deaminase of Escherichia coli, the crystal structure of which is known. The model has structural features shared by aminotransferases, suggesting that tertiary structural elements may be responsible for the transaminating activity observed for rat liver threonine deaminase.

Amino Acid Sequence↗

PubMed2003

Rational design and molecular diversity for the construction of anti-alpha-bungarotoxin antidotes with high affinity and in vivo efficiency.

The structure of peptide p6.7, a mimotope of the nicotinic receptor ligand site that binds alpha-bungarotoxin and neutralizes its toxicity, was compared to that of the acetylcholine binding protein. The central loop of p6.7, when complexed with alpha-bungarotoxin, fits the structure of the acetylcholine binding protein (AChBP) ligand site, whereas peptide terminal residues seem to be less involved in toxin binding. The minimal binding sequence of p6.7 was confirmed experimentally by synthesis of progressively deleted peptides. Affinity maturation was then achieved by random addition of residues flanking the minimal binding sequence and by selection of new alpha-bungarotoxin binding peptides on the basis of their dissociation kinetic rate. The tetra-branched forms of the resulting high-affinity peptides were effective as antidotes in vivo at a significantly lower dose than the tetra-branched lead peptide.

Amino Acid Sequence↗