Maurizio Paci

The structure of the translational initiation factor IF1 from E.coli contains an oligomer‐binding motif

The structure of the translational initiation factor IF1 from Escherichia coli has been determined with multidimensional NMR spectroscopy. Using 1041 distance and 78 dihedral constraints, 40 distance geometry structures were calculated, which were refined by restrained molecular dynamics. From this set, 19 structures were selected, having low constraint energy and few constraint violations. The ensemble of 19 structures displays a root‐mean‐square deviation versus the average of 0.49 Å for the backbone atoms and 1.12 Å for all atoms for residues 6–36 and 46–67. The structure of IF1 is characterized by a five‐stranded β‐barrel. The loop connecting strands three and four contains a short 310 helix but this region shows considerably higher flexibility than the β‐barrel. The fold of IF1 is very similar to that found in the bacterial cold shock proteins CspA and CspB, the N‐terminal domain of aspartyl‐tRNA synthetase and the staphylococcal nuclease, and can be identified as the oligomer‐binding motif. Several proteins of this family are nucleic acid‐binding proteins. This suggests that IF1 plays its role in the initiation of protein synthesis by nucleic acid interactions. Specific changes of NMR signals of IF1 upon titration with 30S ribosomal subunit identifies several residues that are involved in the interaction with ribosomes.

Syringopeptins, new phytotoxic lipodepsipeptides of Pseudomonas syringae pv. syringae

The primary structure of some new lipodepsipeptides named syringopeptins, produced by plant pathogenic strains of Pseudopmonas syringae pv. syringae has been determined by a combination of chemical methods, 1H and 13C NMR spectroscopy and FAB mass spectrometry. Two syringomycin‐producing strains afforded 3‐hydroxydecanoyl‐Dhb‐Pro‐Val‐Val‐Ala‐Ala‐Val‐Val‐Dhb‐Ala‐Val‐Ala‐Ala‐Dhb‐aThr‐Ser‐Ala‐Dhb‐Ala‐Dab‐Dab‐Tyr, with Tyr acylating a Thr to form a macrolactone ring, and smaller amounts of the 3‐hydroxydodecanoyl homologue. Evidence was obtained that a third syringomycin‐producing strain and a syringotoxin‐producing strain synthesize 3‐hydroxydecanoyl‐Dhb‐Pro‐Val‐Ala‐Ala‐Val‐Leu‐Ala‐Ala‐Dhb‐Val‐Dhb‐Ala‐Val‐Ala‐Ala‐Dhb‐aThr‐Ser‐Ala‐Val‐Ala‐Dab‐Dab‐Tyr, with Tyr and aThr forming again the macrolactone ring, and smaller amounts of the 3‐hydroxydodecanoyl homologue.

Structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae

The covalent structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae isolates, pathogenic on various species of citrus trees, has been deduced from ID and 2D 1H‐ and 13C‐NMR spectra combined with extensive FAB‐MS data and results of some chemical reactions. Similarly to syringomicins and syringostatins, produced by other plant pathogenic strains of P. syringae pv. syringae, syringotoxin is a lipodep‐sinonapeptide. Its peptide moiety corresponds to Ser‐Dab‐Gly‐Hse‐Om‐aThr‐Dhb‐(3‐OH)Asp‐(4‐Cl)Thr with the terminal carboxy group closing a macrocyclic ring on the OH group of the N‐terminal Ser, which in turn is N‐acetylated by 3‐hydroxytetradecanoic acid.

Novel bioactive lipodepsipeptides from Pseudomonas syringae: The pseudomycins

The covalent structure and most of the stereochemistry of the pseudomycins, bioactive metabolites of a transposon‐generated mutant of a Pseudomonas syringae wild‐type strain proposed for the biological control of Dutch elm disease, have been determined. While two pseudomycins are identical to the known syringopeptins 25‐A and 25‐B, pseudomycins A, B, C, C′ are new lipodepsinonapeptides. For all of these the peptide moiety corresponds to l‐Ser‐d‐Dab‐l‐Asp‐l‐Lys‐l‐Dab‐l‐aThr‐Z‐Dhb‐l‐Asp(3‐OH) ‐l‐Thr(4‐Cl) with the terminal carboxyl group closing a macrocyclic ring on the OH group of the N‐terminal Ser. This is in turn N‐acylated by 3,4‐dihydroxytetradecanoate in pseudomycin A, by 3‐hydroxytetradecanoate in pseudomycin B, by 3,4‐dihydroxyhexadecanoate in pseudomycin C, and by 3‐hydroxyhexadecanoate in pseudomycin C′. Some preliminary data on the biological activity of pseudomycin A are reported.

Corceptins, new bioactive lipodepsipeptides from cultures of Pseudomonas corrugata

© 1998 Federation of European Biochemical Societies.

A kinetic model for the internal motions of proteins: Diffusion between multiple harmonic wells

The dynamics of collective protein motions derived from Molecular Dynamics simulations have been studied for two small model proteins: initiation factor I and the B1 domain of Protein G. First, we compared the structural fluctuations, obtained by local harmonic approximations in different energy minima, with the ones revealed by large scale molecular dynamics (MD) simulations. It was found that a limited set of harmonic wells can be used to approximate the configurational fluctuations of these proteins, although any single harmonic approximation cannot properly describe their dynamics.

Contryphan-Vn: a modulator of Ca2+-dependent K+ channels ☆

Contryphan-Vn is a D-tryptophan-containing disulfide-constrained nonapeptide isolated from the venom of Conus ventricosus, the single Mediterranean cone snail species. The structure of the synthetic Contryphan-Vn has been determined by NMR spectroscopy. Unique among Contryphans, Contryphan-Vn displays the peculiar presence of a Lys-Trp dyad, reminiscent of that observed in several voltage-gated K(+) channel blockers. Electrophysiological experiments carried out on dorsal unpaired median neurons isolated from the cockroach (Periplaneta americana) nerve cord on rat fetal chromaffin cells indicate that Contryphan-Vn affects both voltage-gated and Ca(2+)-dependent K(+) channel activities, with composite and diversified effects in invertebrate and vertebrate systems. Voltage-gated and Ca(2+)-dependent K(+) channels represent the first functional target identified for a conopeptide of the Contryphan family. Furthermore, Contryphan-Vn is the first conopeptide known to modulate the activity of Ca(2+)-dependent K(+) channels.

ATP Acts as a Regulatory Effector in Modulating Structural Transitions of Cytochrome c: Implications for Apoptotic Activity†

The binding of lipids (free fatty acids as well as acidic phospholipids) to cytochrome c (cyt c) induces conformational changes and partial unfolding of the protein, strongly influencing cyt c oxidase/peroxidase activity. ATP is unique among the nucleotides in being able to turn non-native states of cyt c back to the native conformation. The peroxidase activity acquired by lipid-bound cyt c turns out to be very critical in the early stages of apoptosis. Nucleotide specificity is observed for apoptosome formation and caspase activation, the cleavage occurring only in the presence of dATP or ATP. In this study, we demonstrate the connection between peroxidase activity and oleic acid-induced conformational transitions of cyt c and show how ATP is capable of modulating such interplay. By NMR measurement, we have demonstrated that ATP interacts with a site (S1) formed by K88, R91, and E62 and such interaction was weakened by mutation of E62, suggesting the selective role in the interaction played by the base moiety. Interestingly, the interactions of ATP and GTP with cyt c are significantly different at low nucleotide concentrations, with GTP being less effective in perturbing the S1 site and in eliciting apoptotic activity. To gain insights into the structural features of cyt c required for its pro-apoptotic activity and to demonstrate a regulatory role for ATP (compared to the effect of GTP), we have performed experiments on cell lysates by using cyt c proteins mutated on amino acid residues that, as suggested by NMR measurements, belong to S1. Thus, we provide evidence that ATP acts as an allosteric effector, regulating structural transitions among different conformations and different oxidation states of cyt c, which are endowed with apoptotic activity or not. On this basis, we suggest a previously unrecognized role for ATP binding to cyt c at low millimolar concentrations in the cytosol, beyond the known regulatory role during the oxidative phosphorylation in mitochondria.

The Influence of a Sports Drink on the Postexercise Metabolism of Elite Athletes as Investigated by NMR-Based Metabolomics

Objective: The aim of this study is to evaluate the systemic effects of an isotonic sports drink on the metabolic status of athletes of the Italian Olympic rowing team during recovery after strenuous and prolonged physical exercise by means of nuclear magnetic resonance (NMR)-based metabolomics analysis on plasma and urine. Methods: Forty-four male athletes of the Italian Olympic rowing team were enrolled in a double-blind crossover study. All subjects underwent 2 evaluations at 1-week intervals. The evaluation was performed on a rowing ergometer after strenuous physical exercise to produce a state of dehydration. Afterward, the athletes were rehydrated either with a green tea–based carbohydrate-hydroelectrolyte drink or with oligomineral water. Three blood samples were drawn for each subject: at rest, after the exercise, and following rehydratation, while 2 urine samples were collected: at rest and after the rehydratation period. Biofluid samples were analyzed by high-resolution 1H NMR metabolic profiling combined with multilevel simultaneous data-analysis (MSCA) and partial-least squares-discriminant analysis (PLS-DA). Results: The between-subject variations, as evaluated by MSCA, reflected the variations of lactate levels induced by the physical exercise. Analysis of the within-individual variance using multilevel PLS-DA models of plasma and urine metabolic profiles showed an effect of the green tea–based sports drink on glucose, citrate, and lactate levels in plasma and on acetone, 3-OH-butyrate, and lactate levels in urine. The increase of caffeine and hippuric acid levels in urine indicated the absorption of green tea extract components. Conclusions: NMR-based metabolomics allowed the complex effects of a green tea extract–based carbohydrate/hydroelectrolyte beverage on the energy metabolism of athletes during recovery by postexercise rehydration to be evaluated.

Transglutaminase crosslinking and structural studies of the human small proline rich 3 protein

The cell envelope (CE) is a vital structure for barrier function in terminally differentiated dead stratified squamous epithelia. It is assembled by transglutaminase (TGase) cross-linking of several proteins, including SPR3 in certain specialized epithelia normally subjected to mechanical trauma. We have expressed recombinant human SPR3 in order to study its cross-linking properties. It serves as a complete substrate for, and is cross-linked at similar efficiencies by, the three enzymes (TGases 1, 2 and 3) that are widely expressed in many epithelia. Multiple adjacent glutamines (4, 5, 16, 17, 18, 19 and 167) and lysines (6, 21, 164, 166 and 168) of only head and tail domain sequences are used for cross-linking. However, each enzyme preferentially uses certain residues on the head domain. Moreover, our in vitro data suggest a defined temporal order of cross-linking of SPR3 in vivo: It is first cross-linked by TGase 3 into short intra- and inter-chain oligomers which are later further cross-linked to the CE by TGase 1. To investigate the absence of cross-linking in the central domain (e.g. lysine in position 2 of each of the 16 repeats) we performed structural studies on recombinant SPR3 and on a synthetic peptide containing three repeats of the central domain. 2D H-1 NMR spectroscopy, TOCSY and ROESY, shows strong and medium intensity NOEs connectivities along the amino acid sequence with one weak long range NOE contact between Thr and Cys of subsequent repeats. Distance geometry computation on the basis of intensities of NOEs found generated 50 compatible structures grouped in three main families differing by the number of H-bonds. These measurements were repeated at different concentrations of trifluoroethanol (TFE)-water mixture, an α-helical promoting solvent, in order to check the stability of the conformations determined; no changes were observed up to 50% TFE in solution. Also temperature changes did not produce any variation in the ROESY spectrum in the same condition as above. The NMR and circular dichroism data strongly indicate the presence of an ordered (not α-helix nor β-sheet) highly flexible structure in the eight amino acids repetitive units of SPR3, confirming the prediction of one possible β-turn per each repeating unit. Thus, biochemical and biophysical data, strongly support SPR3 to function as a flexible cross-bridging protein to provide tensile strength or rigidity to the CE of the stratified squamous epithelia in which it is expressed.