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M Saviano

Publications and source records attributed to M Saviano.

At least 37 records · Page 2Linked to original sources
PubMed2000

Solid state structural analysis of the cyclooctapeptide cyclo- (Pro1-Pro-Phe-Phe-Ac6c-Ile-D-Ala-Val8).

A solid state analysis of the cyclic octapeptide c(-Pro(1)-Pro-Phe-Phe-Ac(6)c-Ile-D-Ala-Val(8)-) (C8-CLA), containing the Pro-Pro-Phe-Phe sequence, followed by the bulky helicogenic C(alpha,alpha)-dialkylated 1-aminocyclohexane-1-carboxylic acid (Ac(6)c) residue and a D-Ala residue in position 7, has been carried out by x-ray diffraction. The crystals, grown from a DMSO solution, are monoclinic, space group P2(1) with a = 13.458(3) A, b = 19. 404(5) A, c = 21.508(4) A, and beta = 90.83(6) degrees, with two independent cyclic molecules in the asymmetric unit, two DMSO molecules, and three water molecules. The structure has been solved using the half and bake procedure by Sheldrick, and refined to final R1 and wR2 indices of 0.0613 and 0.1534 for 9867 reflections with I > 2sigma(I). This cyclic peptide, a deletion analogue of the naturally occurring cyclic nonapeptide cyclolinopeptide A [c(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val), CLA] has been designed to study the influence of the ring size reduction on the conformational behavior of CLA and more in general to obtain structural information on asymmetric cyclic octapeptides. The compound exhibits, in the solid state, a "banana-twisted" conformation with a cis peptide bond located between the two proline residues. Five intramolecular H bonds stabilize the structure: one type VIa beta-turn, two consecutive type III/I beta-turns, one gamma-turn, and one C(16) bend. The structure has also been compared with either the solution structure previously reported by us and obtained by nmr and computational analysis, and with solid state structural data reported in the literature on cyclic octapeptides.

Amino Acid Sequence↗
PubMed2000

Conformational restriction through C alpha i <--> C alpha i cyclization: Ac12c, the largest cycloaliphatic C alpha,alpha- disubstituted glycine known.

Two complete series of N-protected, monodispersed oligopeptide esters to the pentamer level from 1-aminocyclododecane-1-carboxylic acid (Ac(12)c), an alpha-amino acid conformationally constrained through C(alpha)(i) <--> C(alpha)(i) cyclization, and either L-Ala or Aib residues, along with the N-protected Ac(12)c homopeptide alkylamide series from monomer to trimer, have been synthesized by solution methods and fully characterized. The solution-preferred conformations of these peptides have been assessed by Fourier transform ir absorption and (1)H-nmr techniques. Moreover, the molecular structures of one derivative (Z-Ac(12)c-OH) and three peptides [the tripeptide ester Z-L-Ala-Ac(12)c-L-Ala-OMe, the tripeptide alkylamide Z-(Ac(12)c)(3)-NHiPr, and the tetrapeptide ester Z-(Aib)(2)-Ac(12)c-Aib-OtBu (Aib, alpha-aminoisobutyric acid)] have been determined in the crystal state by x-ray diffraction. The results obtained point to the conclusion that beta-bends and 3(10)-helices are preferentially adopted by peptides based on Ac(12)c, the largest cycloaliphatic C-disubstituted glycine known. A comparison with the structural tendencies extracted from published works on peptides from Aib, the prototype of C-disubstituted glycines, and the other extensively studied members of the class of 1-aminocycloalkane-1-carboxylic acids (Ac(n) c, with n = 3-9), is made and the implications for the use of the Ac(12)c residue in the Ac(n) c scan approach of conformationally restricted analogues of bioactive peptides are briefly discussed.

Alanine↗
PubMed2000

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).

Amino Acid Sequence↗
PubMed2000

Preferred conformation of peptides based on cycloaliphatic C(alpha,alpha)-disubstituted glycines: 1-amino-cycloundecane-1-carboxylic acid (Ac11c).

Two complete series of N-protected oligopeptide esters to the pentamer level from 1-amino-cycloundecane-1-carboxylic acid (Ac11c), an alpha-amino acid conformationally constrained through a medium-ring C(i)alpha<-->C(i)alpha cyclization, and either the L-Ala or Aib residue, along with the N-protected Ac11c monomer and homo-dimer alkylamides, have been synthesized by solution methods and fully characterized. The preferred conformation of these model peptides has been assessed in deuterochloroform solution by FT-IR absorption and 1H-NMR techniques. Furthermore, the molecular structures of one derivative (Z-Ac11c-OH) and two peptides (the tripeptide ester Z-Aib-Ac11c-Aib-OtBu and the pentapeptide ester Z-Ac11c-(Aib)2-Ac11c-Aib-OtBu) have been determined in the crystal state by X-ray diffraction. The experimental results support the view that beta-bends and 3(10)-helices are preferentially adopted by peptides rich in Ac11c, the second largest cycloaliphatic C(alpha,alpha)-disubstituted glycine known. This investigation has allowed the authors to approach the completion of a detailed conformational analysis of the whole 1-amino-cycloalkane-1-carboxylic acid (Ac(n)c, with n = 3-12) series, which represents the prerequisite for their recent proposal of the 'Ac(n)c scan' concept.

Acids, Carbocyclic↗
PubMed2000

Computational procedures to explain the different biological activity of DNA/DNA, DNA/PNA and PNA/PNA hybrid molecules mimicking NF-kappaB binding sites.

Peptide nucleic acids (PNA) have recently been proposed as alternative reagents in experiments aimed to the control of gene expression. In PNAs, the pseudopeptide backbone is composed of N-(2-aminoethyl)glycine units and therefore is stable in human serum and cellular extracts. PNAs hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, forming Watson-Crick double helices and giving rise to highly stable (PNA)2-RNA triplexes with RNA targets. Therefore, antisense and antigene PNAs have been synthetized and characterized. The major issue of the present paper is to describe some computational procedures useful to compare the behaviour of PNA double stranded molecules and PNA/DNA hybrids with the behaviour of regular DNA duplexes in generating complexes with DNA-binding proteins. The performed computational analyses clearly allow to predict that the lack of charged phosphate groups and the different shape of helix play a critical role in the binding efficiency of NF-kappaB transcription factors. These computational analyses are in agreement with competitive gel shift and UV-cross linking experiments. These experiments demonstrate that NF-kappaB PNA/PNA hybrids do not interact efficiently with proteins recognizing the NF-kappaB binding sites in genomic sequences. In addition, the data obtained indicate that the same NF-kappaB binding proteins recognize both the NF-kappaB DNA/PNA and DNA/DNA hybrids, but the molecular complexes generated with NF-kappaB DNA/PNA hybrids are less stable than those generated with NF-kappaB target DNA/DNA molecules.

Base Sequence↗
PubMed2000

[Surgical treatment of hemorrhoidal disease using circular mechanical stapler. Analysis of costs].

BACKGROUND: An analysis of surgical treatment costs of haemorrhoid disease with the use of a new circular stapler, is made comparing this operation with Milligan Morgan's technique. The money and management saving due to the reduction of operation time and postoperative stay compensates present costs due to stapler. METHODS: 35 uniform patients (number, age, sex, grade of haemorrhoid disease and surgical equipe) are considered and divided into two groups of study to evaluate perspectively the surgical costs. RESULTS: The cost of treatment with stapler per patient is like Milligan Morgan's treatment (1.714.681 lire versus 1.681.893), with an important management saving of postoperative days (16 hours versus 42 hours). CONCLUSIONS: Moreover there are psychologic and social advantages, not quantifiable but considerable, due to the early physical recovery and to the absence of out-patient dressing cycles peculiar of this surgical technique, with a rapid social and working reinstatement of patients (4-5 days versus 4-5 weeks of conventional intervention).

Adult↗
PubMed1999

Prediction and experimental testing of Bacillus acidocaldarius thioredoxin stability.

In order to investigate further the determinants of protein stability, four mutants of thioredoxin from Bacillus acidocaldarius were designed: K18G, R82E, K18G/R82E, and D102X, in which the last four amino acids were deleted. The mutants were constructed on the basis of molecular dynamic studies and the prediction of the structure of thioredoxin from B. acidocaldarius, performed by a comparative molecular modelling technique using Escherichia coli thioredoxin as the reference protein. The mutants obtained by PCR strategy were expressed in E. coli and then characterized. CD spectroscopy, spectrofluorimetry and thermodynamic comparative studies permitted comparison of the relative physicochemical behaviour of the four proteins with that of the wild-type protein. As predicted for the molecular dynamic analysis at 500 K in vacuo, the wild-type structure was more stable than that of the mutants; in fact the Tm of the four proteins showed a decrease of about 15 degrees C for the double and the truncated mutants, and a decrease of about 12 degrees C for the single mutants. A difference in the resistance of the proteins to denaturants such as guanidine HCl and urea was revealed; the wild-type protein always proved to be the most resistant. The results obtained show the importance of hydrogen bonds and ion pairs in determining protein stability and confirm that simulation methods are able to direct protein engineering in site-directed mutagenesis.

Bacillus↗
PubMed1999

Maroteaux-lamy syndrome: five novel mutations and their structural localization.

Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ASB). Mutation analysis in Maroteaux-Lamy syndrome resulted in the identification of approximately 40 molecular defects underlying a great genetic heterogeneity. Here we report five novel mutations in Italian subjects: S65F, P116H, R315Q, Q503X, P531R; each defect was confirmed by restriction enzyme or amplification refractory mutation system (ARMS) analysis. We also performed a three-dimensional (3-D) structure analysis of the alterations identified by us, and of an additional 22 point mutations reported by other groups, in an attempt to draw helpful information about their possible effects on protein conformation.

Binding Sites↗
PubMed1998

Conformational behaviour of C(alpha,alpha)-diphenylglycine: folded vs. extended structures in DphiG-containing tripeptides.

The crystal structures of three fully protected tripeptides containing the Dphi g residue (C[alpha,alpha]-diphenylglycine) in the central position are reported, namely Z-Gly-Dphi g-Gly-OMe (a), Z-Gly-Dphi g-Aib-OMe (b) and Z-Aib-Dphi g-Aib-OMe (c). The molecular conformations are quite unusual because the Dphi g residue adopts a folded conformation in the 3(10)-helical region when the following residue adopts a folded conformation of opposite handedness (peptides b and c). In contrast, the Dphi g residue adopts the more frequently observed fully extended conformation when the following residue adopts a semi-extended conformation (peptide a). These findings are in agreement with the theoretical calculations on Ac-Dphi g-Aib-NHCH3 and Ac-Aib-Dphi g-NHCH3 also reported in this work.

Glycine↗
PubMed1998

X-ray structures of new dipeptide taste ligands.

The molecular basis of sweet taste was investigated by carrying out the crystal state conformational analysis by X-ray diffraction of the following dipeptide taste ligands: N-3,3-dimethylbutyl-aspartylphenylalanine methyl ester, I (N-DMB-Asp-Phe-OMe), its sodium salt (N-DMB-Asp-Phe-ONa), II, aspartyl-D-2-aminobutyric acid-(S)-alpha-ethylbenzylamide, III (Asp-D-Abu-(S)-alpha-ethylbenzylamide), aspartyl-N'-((2,2,5,5-tetramethylcyclopentanyl)-carbonyl)-(R)- 1,1-diamino-ethane, IV (Asp-(R)-gAla-TMCP), and aspartyl-D-valine-(R)-alpha-methoxymethylbenzyl amide, V (Asp-D-Val-(R)-alpha-methoxymethylbenzylamide). With the exception of the sodium salt II, all compounds are sweet-tasting, showing in some cases considerable potency enhancement with respect to sucrose. The results of this study confirm the earlier model that an 'L-shape' molecular array is essential for eliciting sweet taste for dipeptide-like ligands. In addition, it was established that (i) substitution of the N-terminal group does not inhibit sweet taste, if its zwitterionic character is maintained; (ii) a hydrophobic group located between the stem and the base of the L-shape could be responsible for sweetness potency enhancement, as found in I, III and IV; in fact, the extraordinary potency of the N-alkylated analogue I would support a model with an additional hydrophobic binding domain above the base of the 'L'; (iii) removal of the methyl ester at the C-terminus of compound I with the salt formation gives rise to the tasteless compound II; (iv) for the first time all possible side-chain conformers (g-, g+ and t) for the N-substituted aspartyl residue were observed; and (v) a retro-inverso modification, incorporated at position 2 of the dipeptide chain, confers greater flexibility to the molecule, as demonstrated by the contemporary presence of six conformationally distinct independent molecules in the unit cell and yet sweet taste properties are maintained, as found in IV.

Computer Simulation↗
PubMed1998

Structure and dynamics of elastin building blocks. Boc-LG-OEt, Boc-VGG-OH.

Short di- and tripeptides such as Boc-LG-OEt, Boc-VG-OEt and Boc-VGG-OH, corresponding to abundant repetitive sequences in elastin, have been extensively studied both in solid state, by X-ray diffraction, and in solution by circular dicroism and nuclear magnetic resonance. Furthermore, theoretical procedures such as simulated annealing and molecular dynamics were also performed on these peptides. In general, the results indicate that no one single structure (be folded or extended) could be representative for these sequences in the protein, but rather that a multiplicity of interconverting conformers, ranging from folded to extended structures, should be considered. In any case, these structures, e.g. beta-turns, polyglycine II and beta-conformations, are those previously suggested to participate to conformational equilibria of elastin.

Circular Dichroism↗
PubMed1998

Computational analysis of the thermal stability in thioredoxins: a molecular dynamics approach.

The knowledge of the relationship between the three-dimensional structure of a protein and its biological and stability is one of the most challenging problem in protein chemistry, since offers the possibility of changing both the specific action of a protein and its stability. In this work, we have approached the problem with studies on a protein family, the thioredoxins, using homology procedures, molecular dynamics simulations in vacuo at 300 K and 500 K and in water solution at 300 K, to determine the relationship between the three-dimensional structure of these proteins and their thermal stability. A comparative analysis, using computational approach, was performed between two thioredoxins with different resistance to temperature. Results obtained using the molecular dynamics techniques and minimization procedures give explanations of the experimental data, underlining that these techniques are able to correlate the increase in protein stabilization with the conformational and structural changes caused by single amino acid replacement. In addition, we report the factors that can be used as a guide in protein engineering and in site-directed mutagenesis to increase or decrease thermal stabilization for this protein family.

Amino Acid Sequence↗
PubMed1998

Specific interaction between bovine cyclophilin A and synthetic analogues of cyclolinopeptide A.

Like cyclosporin A, cyclolinopeptide A binds specifically bovine cyclophilin A, inhibiting its peptidyl-prolyl cis-trans isomerase activity. We describe here the protein interaction with several synthetic analogues of cyclolinopeptide A, which are either homodetic or disulphide bridged heterodetic cyclopeptides characterized by different ring dimensions, in terms of dissociation and inhibition constants evaluated by fluorescence and inhibition of the enzyme activity, respectively. Dissociation constants from fluorescence experiments are practically identical and about 20-fold lower than for cyclosporin A. On the other hand, inhibition constants differ from compound to compound and are higher than for cyclosporin A. This result is therefore difficult to rationalize, but we would suggest decoupling between binding and inhibitory ability of cyclopeptides. The Pro1 residue of cyclolinopeptide A seems to play a fundamental role in determining the inhibition of the rotamase activity of cyclophilin A, as the homodetic analogue lacking this residue does not show any inhibitory ability. Similarly, heterodetic analogues with a ring size smaller than 7 residues do not display inhibition. We presume that the sequence -Pro-Pro-Phe-Phe- and a ring size of 8 residues for homodetic cyclic peptides could be used as starting points in the targeted synthesis of cyclopeptides able to bind both cyclosporin A and calcineurin. The only peptide showing similar values of the dissociation and inhibition constant is cyclolinopeptide A. This compound can be considered a novel model for the molecular design of immunosuppressant drugs.

Amino Acid Sequence↗
PubMed1998

Structure of cyclic peptides: the crystal and solution conformation of cyclo(Phe-Phe-Aib-Leu-Pro).

A solid-state and solution conformation analyses of the cyclopentapeptide cyclo(Phe-Phe-Aib-Leu-Pro) has been carried out by X-ray diffraction and nuclear magnetic resonance techniques. The structure of the hexagonal crystals, grown from a methanol solution [a = b = 16.530(4) A, c = 21.356(9) A, space group P6(5), Z = 6], shows the presence of one intramolecular N-H ..O=C hydrogen bond with the formation of a gamma-turn (C7). The Aib3 residue, at the center of the gamma-turn, presents unexpected values of the torsion angles [phi = 70.5 degrees and psi = -73.8 degrees], which have been observed only once before for this helicogenic residue. A cis peptide bond occurs between Leu4 and Pro5; all other peptide bonds are trans. The overall conformation for the cyclopentapeptide with one cis-peptide bond on one side and an intramolecular gamma-turn on the opposite side results in an equatorial topology of the side-chains of the Phe1, Phe2 and Leu4 residues. Indeed, the Calpha-Cbeta and Cbeta-Cgamma bonds of these residues lie approximately in the mean plane of the cyclic ring system. The structure is compared with data in the literature on cyclic pentapeptides. In addition the Pro-Phe-Phe moiety shows a conformation similar to that observed in other larger cyclic bioactive peptides, which indicates a reduced number of conformations for this sequence. The solution study was carried out in three different solvent systems: chloroform, acetonitrile and methanol in the temperature interval 220-300 K. In all three solvents the room temperature spectra show that the peptide is conformationally nonhomogeneous. In acetonitrile at low temperatures it is possible to reduce the conformational equilibrium to two predominant conformers which differ for the cis-trans isomerism of the Leu4-Pro5 peptide bond.

Magnetic Resonance Spectroscopy↗
PubMed1998

[Phantom rectum after Miles' operation].

The authors observed symptoms relating to the existence of a phantom rectum, a phenomenon about which little is still known, in 233 cases (32.1%) in a series of 724 patients undergoing amputation of the abdomino perineal rectum due to malignant neoplasm. The genesis of this phenomenon is attributed to an abnormal reactivity of the cortical and thalamic projection areas with greater response to stimuli from the stump or aspecific visceral stimuli, or even stimuli from the adjacent cortical regions. The psychodynamic aspects linked to the patient's need to develop a different body awareness should not be underestimated; this has to integrate a new anatomic reality, such as colostomy in the place of the anorectum, entailing the relinquishment of the previous body scheme. Prevention and treatment of phantom rectum, which is painful in 27% of cases, is essential for a correct operating technique, adequate psychotherapeutic support, the patient's active participation in a reeducation programme for colostomy management. It should not be forgotten that painful phantom rectum, especially with late onset, may be diagnostically significant as an indicator of the recurrence of neoplastic pathology, sometimes allowing diagnosis to be anticipated by several months. This underlines the importance of carrying out a careful postoperative clinical, biohumoral and radiological control in these patients, including pelvic CT. In the authors' experience of 233 patients suffering from phantom rectum, it was possible to document the presence of neoplastic recidivation in 30 cases (12.8%) using pelvic CT. In these cases, symptoms appeared after a disease-free interval of 26.3 months (range 3-75 months), whereas the presence of phantom rectum without recidivation is usually described in the first 2 months after surgery.

Abdomen↗
PubMed1996

Synthesis and structural characterization of 6I,6II-diamino-6I,6II-dideoxy-cyclomaltoheptaose, a difunctionalized beta-cyclodextrin.

6I,6II-Diamino-6I,6II-dideoxy-cyclomaltoheptaose was prepared using the regioselective procedure described by Tabushi. The difunctionalized beta-cyclodextrin crystallizes as hexadecahydrate in the orthorhombic space group P2(1)2(1)2(1), with a = 11.395(3), b = 32.989(9), c = 17.560(5) A, V = 6601 A3, Z = 4. The structure was solved by molecular replacement techniques using the program PATSEE and was refined to a conventional final R = 0.058 for the 5031 observed reflections with I > or = 3 sigma(I). The beta-CD macrocycle presents only slight differences with respect to uncomplexed hydrated or methylated beta-CD. The macrocycle structure maintains an approximate seven-fold symmetry. The round shape of the cyclodextrin ring is stabilized by intramolecular O-H ... O H-bonds between the secondary hydroxyl groups of neighbouring glucose residues. Along the a axis, the beta-CD molecules are arranged in columns; the macrocycles form a herring-bone pattern, so that the cavity of each beta-CD molecule is closed at each end by neighbouring molecules. The macrocycles are directly linked to each other by H-bonds involving either primary and secondary hydroxyl or amino groups of symmetry-related molecules. The resulting layers are connected to each other by a dense intermolecular hydrogen-bond network, in which solvent molecules participate.

Carbohydrate Sequence↗
PubMed1996

Solution and solid state structure of an aib-containing cyclodecapeptide inhibiting the cholate uptake in hepatocytes.

The conformational analysis of synthetic cyclodecapeptide c(Pro-Phe-phe-Aib-Leu)2 related to the cyclolinopeptide A, in the solid state and solution, has been carried out by x-ray diffraction and nmr spectroscopy. The structure of the monoclinic form obtained from methanol [a = 11.351 (5) A, b = 27.455 (2) A, c = 12.716 (8) A, beta = 99.65 (3) degrees; space group P2(1); Z = 2] shows the presence of six intramolecular NH...CO hydrogen bonds, with formation of four turns (three of type I and one of type III) and two C16 ring structures. All peptide units are trans. The solution structure, as found by nmr, indicates that, at room temperature, the peptide is conformationally homogeneous; the structure determined is perfectly symmetrical and topologically similar to that found in the solid state. The cyclodecapeptide exhibits similar biological activity to cyclolinopeptide A.

Animals↗