NMR studies on the surface accessibility of the archaeal protein Sso7d by using TEMPOL and Gd(III)(DTPA-BMA) as paramagnetic probes.

Understanding how proteins are approached by surrounding molecules is fundamental to increase our knowledge of life at atomic resolution. Here, the surface accessibility of a multifunctional small protein, the archaeal protein Sso7d from Sulfolobus solfataricus, has been investigated by using TEMPOL and Gd(III)(DTPA-BMA) as paramagnetic probes. The DNA binding domain of Sso7d appears very accessible both to TEMPOL and Gd(III)(DTPA-BMA). Differences in paramagnetic attenuation profiles of (1)H-(15)N HSQC protein backbone amide correlations, observed in the presence of the latter paramagnetic probes, are consistent with the hydrogen bond acceptor capability of the N-oxyl moiety of TEMPOL to surface exposed Sso7d amide groups. By using the gadolinium complex as a paramagnetic probe a better agreement between Sso7d structural features and attenuation profile is achieved. It is interesting to note that the protein P-loop region, in spite of the high surface exposure predicted by the available protein structures, is not approached by TEMPOL and only partially by Gd(III)(DTPA-BMA).

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.

Structurally driven selection of human hepatitis C virus mimotopes.

A structural genomics approach is proposed for the development of new diagnostic kits. It combines molecular modelling, peptide synthesis and immunological tests. The preliminary step is the development of a reliable three-dimensional structure of an immunodominant protein of the target pathogenic organism using the various bioinformatic strategies that are now available to structural biologists. Once the protein structure is obtained, the most surface-exposed fragments with minimal sequence variability among the different strains reported in the genomic data bank are reproduced synthetically as linear peptides. These peptides are then tested for immunoreactivity with the plasma of infected patients to determine whether the synthetic molecules have antigenic activity and can therefore be used to detect infecting agents. This structurally driven selection of mimotopes was successfully performed for the human hepatitis C virus, as five peptides that specifically interact with the plasma of HCV-infected patients were identified solely on the basis of the three-dimensional structure predicted for the E2 homodimer of the la viral subtype. A similar approach could easily be extended to a large variety of immunogenic proteins from other pathogenic organisms.

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.

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.

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.

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.

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.

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.

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.

NMR studies on Ni(II) induced cyclization of a histidine-tagged peptide.

A linear decapeptide, HGASYQDLGH, was synthesized and used as a model to evaluate the effect of nickel addition upon non-covalent backbone cyclization. The NMR data, obtained for the peptide in the presence of the metal ion, support the existence of predominant folded structures in solution, where the two His residues are maintained close to each other. These results suggest that insertion of even a single His residue at each peptide terminus can be used efficiently to reduce peptide flexibility without any backbone modification.

Metal ion complexation and folding of linear peptides.

A Linear peptide, GASYQDLG was synthesised and used as a model to evaluate the effects of nickel additions to increase the conformational stability. The NMR data obtained for the peptide and its histidyl derivative (H)(3)GASYQDLG(H)(3) suggest that in solution folded structures are present only for the H-tagged peptide-Ni(II) ion system. These results suggest that metal ions and additions of a double histidine tags of suitable length can be used as efficient tools to reduce peptide flexibility without other internal modifications. Synthesis of H-tagged analogs could offer a promising strategy for large-scale preparation of diagnostic tools and, in general, whenever more rigid molecular structures should be advisable.

Peptide-protein interactions studied by surface plasmon and nuclear magnetic resonances.

The structural features of the complexes that alpha-bungarotoxin forms with three different synthetic peptides, mimotopes of the nicotinic acetylcholine receptor binding site, have been compared to the corresponding nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) data. For the considered peptides, the observed different affinities towards the toxin could not be accounted simply by static structural considerations. A combined analysis of the SPR- and NMR-derived dynamic parameters shows new correlations between complex formation and dissociation and the overall pattern of intramolecular and intermolecular nuclear Overhauser effects. These features could be crucial for a rational design of protein ligands.

NMR structure of alpha-bungarotoxin free and bound to a mimotope of the nicotinic acetylcholine receptor.

A combinatorial library approach was used to produce synthetic peptides mimicking the snake neurotoxin binding site of nicotinic receptors. Among the sequences, which inhibited binding of alpha-bungarotoxin to muscle and neuronal nicotinic receptors, HRYYESSLPWYPD, a 14-amino acid peptide with considerably higher toxin-binding affinity than the other synthesized peptides, was selected, and the structure of its complex with the toxin was analyzed by NMR. Comparison of the solution structure of the free toxin and its complex with this peptide indicated that complex formation induced extensive conformational rearrangements mainly at finger II and the carboxy terminus of the protein. The peptidyl residues P10 and Y4 seemed to be critical for peptide folding and complex stability, respectively. The latter residue of the peptide strongly interacted with the protein by entering a small pocket delimited by D30, C33, S34, R36, and V39 toxin side chains.