The FERMI free-electron lasers.

FERMI is a seeded free-electron laser (FEL) facility located at the Elettra laboratory in Trieste, Italy, and is now in user operation with its first FEL line, FEL-1, covering the wavelength range between 100 and 20 nm. The second FEL line, FEL-2, a high-gain harmonic generation double-stage cascade covering the wavelength range 20-4 nm, has also completed commissioning and the first user call has been recently opened. An overview of the typical operating modes of the facility is presented.

Correlation between fluorescence and structure in the orange-emitting GFP-like protein, monomeric Kusabira Orange.

The mKO is the monomeric version of Kusabira Orange, a GFP-like protein emitting bright orange fluorescence at 559 nm. This protein shows the characteristic ß-barrel motif typical of the fluorescent protein family which it belongs to, similar spectral properties to the tetrameric form and an exceptional photo-stability to pH changes. Here, we demonstrate that mKO in solution at physiological pH exhibits a secondary structure analogue to that of the crystal. Moreover, we describe the thermal unfolding, revealing an outstanding structural stability with a denaturation temperature close to 90 °C and identifying the existence of a thermodynamic intermediate. The denaturation process of mKO results to be absolutely irreversible because of the complete lost of the native structure and the consequent aggregation, while the presence of the intermediate state is most likely due to coexistence of two different species of mKO, with protonated and deprotonated chromophore respectively, that affects the fluorescence properties and the structural stability of the protein.

Assessing terminal restriction fragment length polymorphism suitability for the description of bacterial community structure and dynamics in hydrocarbon-polluted marine environments.

The distribution of bacterial communities terminal restriction fragment length polymorphism (T-RFLP) fingerprint patterns was evaluated at three proximal hydrocarbon-contaminated sites located within the harbour of Messina. In order to analyse the short-term variability of the individual terminal restriction fragment (T-RF) patterns, water samples were collected at the three sites on three occasions within 3 months (T(0), T(90) and T(91)). Four sample sizes, from 50 to 1000 ml for each collected sample, were analysed separately (36 total analysed samples) to evaluate the relationship between the sample size and the bacterial diversity estimates. The dominant T-RF groups mostly belonged to signatures of putative hydrocarbon-degrading bacteria, as revealed by the virtual analysis of the obtained bands. In order to test whether significant differences were occurring between the analysed samples, the Kruskal-Wallis non-parametric test was applied to the T-RF data set. Neither significant influence of the sample size nor short spatial variability within the three sampled sites was detected for each sampling time. On the contrary, significant temporal changes in the diversity of the bacterial communities were observed. These results were confirmed by the non-metric multidimensional scales (nMDS) analysis of the whole set of samples, which indicated three main groups corresponding to the three different sampling times. In summary, the T-RFLP technique, although a polymerase chain reaction-based method, proved to be a suitable technique for monitoring polluted marine environments, typically characterized by low diversity and high relative abundances of a few dominant groups.

Search for the prewave zone effect in transition radiation.

Results of the first experimental search for the effect of the prewave zone in near-infrared transition radiation are presented. A substantial difference in the spatial distribution of transition relation for two different wavelengths (450 nm and 1600 nm) was observed. Experimental data are in a good, though not complete, agreement with the theory.

A type-II beta-turn, proline-containing, cyclic pentapeptide as a building block for the construction of models of the cleavage site of pro-oxytocin.

Previous studies have indicated that proteolytic activation of pro-hormones and pro-proteins occurs most frequently at the level of basic amino acids arranged in doublets and that the dibasic sites are situated in or next to beta-turns. Investigations utilizing synthetic peptides reproducing the N-terminal processing domain of pro-oxytocin-neurophysin have suggested a close relationship between the secondary structure of the cleavage locus and enzyme recognition, the minimal recognized sequence being the -Pro-Leu-Gly-Gly-Lys-Arg-Ala-Val-Leu- segment of the native precursor. NMR investigations and energy minimization studies have demonstrated that this sequence is organized in two type-II beta-turns involving the -Pro-Leu-Gly-Gly- and -Lys-Arg-Ala-Val- sequences. To further strengthen the above reported hypothesis and to study the role of turn subtypes, a new proline containing cyclic substrate of the processing enzyme, in which the N-terminal side that comes before the Lys-Arg pair is constrained to adopt a type-lI beta-turn, has been synthesized. The presence of a type-II beta-turn structure in this cyclic peptide model has been demonstrated by a combined NMR, CD and FT-IR absorption investigation. A preliminary study shows that PC1 is able to recognize and process our constrained substrate.

Solid state and solution conformation of [Ala7]-phalloidin: a synthetic phallotoxin analogue.

Phallotoxins are toxic compounds produced by poisonous mushroom Amanita phalloides and belong to the class of bicyclic peptides with a transannular thioether bridge. Their intoxication mechanism in the liver involves a specific binding of the toxins to F-actin that, consequently, prevents the depolymerization equilibrium with G-actin. Even though the conformational features of phallotoxins have been worked out in solution, the exact mechanism of interaction with F-actin is still unknown. In this study a toxic phalloidin synthetic derivative, bicyclo(Ala1-D-Thr2-Cys3-cis-4-hydroxy-Pro4-Ala5-2-mercapto-Trp6-Ala7)(S-3-->6) has been synthesized. A substitution at position 7. with an Ala residue replaces the 4,5-dihydroxy-Leu present in the natural phalloidin. This analogue has formed crystals suitable for X-ray analysis, and represents the first case for such a class of compounds. The solid-state structure as well as the solution conformation have been evaluated. NMR techniques have been used to extract interproton distances as restraints in subsequent molecular dynamics calculations. Finally, a direct comparison between structures in solution and in the solid state is presented.

Phalloidin synthetic analogues: structural requirements in the interaction with F-actin.

Synthetic derivatives of phalloidin have been investigated in solution by circular dichroism (CD) and NMR spectroscopy. They differ from natural phalloidin (PHD). bicyclo(Ala1-D-Thr2-Cys3-cis-4-hydroxy-Pro4-Ala5-2-mercapto-Trp6-(OH)2Leu7)(S-3 --> 6), in that they are modified at positions 2, 3, and 7. Among these synthetic analogues, structural differences and varying degrees of atropisomerism are found. By comparing the respective molecular models obtained by restrained molecular dynamics (RMD) simulations based on experimental NMR data, structural features that may be responsible for the different biological behavior become apparent. Our results indicate that the structural changes that result from an inversion of chirality of residue 3 lead to a complete loss of toxicity. Conversely, toxicity is less affected by the structural changes that stem from an inversion of chirality of residue 2. Moreover, unlike the other phallotoxins, when the thioether unit bridges to the opposite face of the main peptide ring, in contrast to the situation in other phallotoxins, large structural changes are observed as well as a total loss of activity. Molecular models of the synthetic phalloidin analogues have been used to investigate the necessary structural requirements for the interaction with F-actin. To this end, the F-actin/PHD model of M. Lorenz et al. was employed; docking experiments of our molecular models in the PHD binding site are presented.

Bicyclic peptides as models of calcium binding sites: synthesis and conformation of a homodetic undecapeptide.

A bicyclic undecapeptide of sequence cyclo-(Ala(1)-Pro(2)-Asp(3)-Glu(4)-Lys(5)-Ala(6)-Pro(7)-Asp(8)-Ser(9) -Glu(10))-cyclo-(10gamma --> 5varepsilon)-Gly(11), designed to mimic the calcium coordination site I of Calmodulin, has been synthesized and its conformation and calcium binding properties have been investigated by means of CD and nmr spectroscopy. The nmr analysis of the free peptide, carried out in DMSO and in TFE/H(2)O at different pH values, shows the presence in solution of one stable conformer, exhibiting trans configuration around both Proline residues. The nmr results in both solvents suggest for the molecule a rectangular shape constituted by two antiparallel beta-strands connected by two beta-turns. Interproton distances, evaluated by NOE contacts, have been used to obtain feasible models by means of Restrained Molecular Dynamic (RMD). The average models from RMD calculations, for both solvents, exhibit good analogies with Calmodulin site I. The model system, when compared with the reference system (Asp(20)-Glu(31) segment in CaM), shows similar dimensions and an effective superimposition of the respective sequence segments Ala(1)-Glu(4) and Thr(28)-Glu(31). The remaining segments of the model peptide exhibit a bending that is intermediate between that of the free and Ca(2+)-coordinated site I. CD spectra, recorded in TFE solutions, point to a 1:1 stoichiometry for the Ca(2+)-peptide complex, with an association constant of at least 1 x 10(5) M(-1).

Conformation and calcium-binding properties of a bicyclic nonapeptide.

The conformation and calcium binding properties of the bicyclic nonapeptide BCP2, cyclo-(Glu(1)-Ala(2)-Pro(3)-Gly(4)-Lys(5)-Ala(6)-Pro(7)-Gly(8))-cyclo-(1gamma --> 5epsilon) Gly(9), have been investigated by means of NMR spectroscopy. Interproton distances, evaluated by nuclear Overhauser effect (NOE) contacts, and straight phi angles, from (3)J(NH-alphaCH), have been used to obtain a feasible model for the BCP2-Ca(2+) (BCP: bicyclic peptide) complex by means of restrained molecular dynamics (RMD). The NMR analysis of the free peptide, carried out in CD(3)CN, shows the presence in solution of at least four conformers in intermediate exchange rate. The addition of calcium ions caused the appearance of a new set of resonances, differing from those observed for the free BCP2. A comparison with published data about the conformational behavior of the closely analogous peptide BCP3, differing from BCP2 for two Leu residues instead of two Ala residues in positions 2 and 6, shows that this simple substitution dramatically increases the peptide flexibility. On the contrary, upon calcium ion addition, both BCP2 and BCP3 reach a strictly close conformation, as strongly testified by the almost identical (1)H-NMR spectra exhibited by both peptides. The RMD molecular model of the BCP2-Ca(2+) complex, here reported, is a quite symmetric structure, presenting a three-dimensional cavity ideal for the binding of spherical cations. Four carbonyls from the main ring (Ala(2), Gly(4), Ala(6) and Gly(8)) point toward it, offering, together with the two carbonyls of the peptide bridge (Gly(9) and gammaGlu(1)), putative coordinations to the cation.

A conformational study in solution of pro-somatostatin fragments by NMR and computational methods.

The results of a conformational study by nuclear magnetic spectroscopy and computational methods on a series of point-mutated synthetic peptides, containing 14 amino acid residues and mimicking the region containing the Arg-Lys dibasic cleavage site of pro-somatostatin, have confirmed the possible role of a well defined secondary structure in the recognition phenomenon by processing enzymes. The importance of the residues located near the Arg-Lys dibasic site in the C-terminal region of the pro-hormone for the cleavage of the precursor into somatostatin-14 has been confirmed. The present structural analysis indicates the occurrence of two beta-turns in the 4-7 and 11-14 regions, flanking the cleavage site, for all the peptides recognized as substrates by the processing enzyme. Interestingly, in the point-mutated analogue not processed by the enzyme and containing the replacement of proline by alanine in position 5 the first -turn is displaced by one residue and involves the Ala5-Arg8 segment. This observation may explain the lack of recognition by the maturation enzyme.

NMR conformational studies on a synthetic peptide reproducing the [1-20] processing domain of the pro-ocytocin-neurophysin precursor.

The combined use of several nuclear magnetic resonance and restrained molecular dynamics techniques allowed the formulation of a molecular model for the preferred solution conformation of a synthetic peptide reproducing the [1-20] processing domain of the pro-ocytocin-neurophysin precursor. In the model, the conformation of the 20-membered tocin ring, with the two Cys1 and Cys6 residues bridged by a disulphide bond, is very close to that observed for isolated ocytocin in the solid state; in addition, a type II beta-turn is postulated for the 7-10 segment of the acyclic tail containing the Lys11-Arg12 processing site, and connecting ocytocin to the neurophysin domain, while the C-terminal 13-20 segment of the molecule is believed to assume a helical structure. This particular structural organization could be important in participating as the favorable conformation for optimal substrate-enzyme active site recognition and processing by specific endoproteases.

Bicyclic peptides as type I/type II beta-turn scaffolds.

We recently reported the rational design, synthetics, and structural characterization of the most potent and selective peptide-based neurokinin A antagonist thus far described: cyclo(Met1-Asp2-Trp3-Phe4-Dap5-Leu6)cyclo(2 beta-5 beta). Its bicyclic structure is characterized by a type I and a type II two beta-turn around Trp3-Phe4 and Leu6-Met1, respectively. In order to understand whether the two different beta-turned structures are determined by the bicyclic structure or by the amino acid type at the corner positions, we have synthesized the pseudo-symmetrical analogue cyclo(Phe1-Asp2-Trp3-Phe4-Dap5-Trp6)cyclo(2 beta-5 beta). The structural characterization in the crystal state and in solution, here reported, gives an experimental evidence that the backbone of the bicyclic structure is a rigid scaffold that can be used to build both a type I and type II beta-turn independently from the amino acid composition.

Design of metal ion binding peptides.

Two cyclic and branched peptides (PLA and AZU) were synthesized with the aim of reproducing the active site of the blue copper proteins plastocyanin and azurin. Both peptides, designed on the basis of the x-ray structures of Poplar plastocyanin and Alcaligenes denitrificans azurin, contain the same coordinating residues of the parent native proteins. The visible spectra of PLA in the presence of equimolar amount of Cu(II) strongly support the interaction between the peptide and copper(II) ion. The CD titration of AZU with the Hg(II) ion indicates for the formation of two species, [AZUHg]+ and [AZUHg2]3+ having binding constants (Keq) of 3.10(6) and 2.10(4) M-1, respectively.

beta-Alanine containing peptides: gamma-turns in cyclotetrapeptides.

In the present paper we describe the synthesis, purification, single-crystal x-ray analysis, solution conformational characterization, and conformational energy calculations of the cyclic tetrapeptide cyclo-(beta-Ala-L-Pro-beta-Ala-L-Val). The peptide was synthesized by classical solution methods and the cyclization of the free tetrapeptide was accomplished in good yields in diluted methylene chloride solution using N,N-dicyclohexyl-carbodiimide. The compound crystallizes in the monoclinic space group P2(1) from ethanol with two independent molecules in the unit cell. All peptide bonds are trans. The nmr molecular conformation in the acetonitrile solution as well as that derived from the molecular dynamic simulation in vacuo is quite different from those observed in the solid state and is very similar to that previously observed for the parent compound cyclo- (beta-Ala-L-Pro-beta-Ala-L-Pro).

Structure and function of protamines: an 1H nuclear magnetic resonance investigation of the interaction of clupeines with mononucleotides.

Protamines form a class of low-molecular-weight proteins that protect the chromosomal DNA in the spermatic cells of eukaryotic organisms. Protamines are located in the small and/or large groove of DNA where they complex the DNA nucleotides. Very little is known up to date on the role and specificity of binding of the various protamine fractions belonging to a single eukaryotic species. In the present paper, a detailed investigation on the complexation properties of the protamine fractions (clupeines) extracted from herrings has been carried out by means of proton nuclear magnetic resonance and ultraviolet absorbtion data. In particular, the binding properties of the clupeine fractions with purinic (5'dAMP) and pyrimidinic (5'dCMP) mononucleotides have been measured and analysed at different clupeine concentrations. The results indicate that, contrary to previous preliminary hypothesis, the three clupeine fractions exhibit quite comparable binding properties toward mononucleotides. In addition it has been found that nucleotides can induce a conformational transition of the disorder-order type in the clupeine molecules and this property is concentration and temperature dependent. It is concluded that, as far as specificity is concerned, the clupeine fractions seem to possess the same behaviour toward mononucleotides.

Model for estimating the refractive-index structure constant in clear-air intermittent turbulence.

We explain discrepancies in comparing estimations of the refractive-index structure constant C(n)(2) in clear air by means of different techniques by taking into account atmospheric intermittency effects. We formulate a model of C(n)(2) in intermittent turbulence on the basis of the Tatarskii theory, and we calculate the mean value of C(n)(2) through a probabilistic approach. We deduce a factor, which gives a measure of the statistical reduction of turbulence that is due to intermittency, within the model framework. A procedure for estimating the mean value of C(n)(2) from data of a specific radiosonde observation is illustrated.

Evidence for the presence of a secondary structure at the dibasic processing site of prohormone: the pro-ocytocin model.

Bioactivation of pro-proteins by limited proteolysis is a general mechanism in the biosynthesis of hormones, receptors and viral protein precursors. This proceeds by cleavage of peptide bonds at the level of single or pairs of basic residues in the proforms. Examination of a number of cleavage loci in various precursors failed to reveal any consensus primary sequence around the dibasic cleavage sites. Thus it has been proposed, on the basis of secondary structure predictions [Rholam, M., Nicolas, P. and Cohen, P. (1986) FEBS Lett., 207, 1-6], that those basic residues which operate as signal loci for the proteolytic enzyme machinery are situated in, or next to, privileged precursor regions most often constituted by flexible and exposed motifs, e.g. beta-turns and/or loops. Peptides reproducing the N-terminal processing domain of the hormone precursor, pro-ocytocin-neurophysin, were examined by a combination of spectroscopical techniques including circular dichroism, infrared Fourier transform and one- and two-dimensional proton NMR. The results indicate that: (i) the region situated on the N terminus of the Lys-Arg doublet is organized as a beta-turn in solution; (ii) the sequential organization of the residues participating in the beta-turn determines the privileged relative orientation of the basic amino acid side chains and the subtype of turn; (iii) the peptide segment situated on the C-terminal side of the dibasic, corresponding to the N-terminal octapeptide of neurophysin, is organized as an alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)