Biomedical subjects

G Saviano

Publications and source records attributed to G Saviano.

At least 19 recordsLinked to original sources

PubMed2001

Solution structure of a sweet protein: NMR study of MNEI, a single chain monellin.

The sweet protein MNEI is a construct of 96 amino acid residues engineered by linking, with a Gly-Phe dipeptide, chains B and A of monellin, a sweet protein isolated from Discoreophyllum cuminsii. Here, the solution structure of MNEI was determined on the basis of 1169 nuclear Overhauser enhancement derived distance restraints and 184 dihedral angle restraints obtained from direct measurement of three-bond spin coupling constants. The identification of hydrogen bonded NH groups was obtained by a combination of H/(2)H exchange data and NH resonance temperature coefficients derived from a series of HSQC spectra in the temperature range 278-328 K. The good resolution of the structure is reflected by the Z-score of the quality checking program in WHAT IF (-0.61). The topology of MNEI, like that of natural monellin and of SCM, another single-chain monellin, is typical of the cystatin superfamily: an alpha-helix cradled into the concave side of a five-strand anti-parallel beta-sheet. The high resolution (14 restraints/residue) 3D structure of MNEI shows close similarity to the crystal structures of natural monellin and of SCM but differs from the solution structure of SCM. The structures of SCM in the crystal and in solution differ in some of the secondary structure elements, but most of all in the relative arrangement of the elements: the four main beta-strands that surround the helix in the crystal structure of SCM, are displaced far from the helix in the solution structure of SCM. These differences were attributed to the fact that SCM is a monomer in solution and a dimer in the crystal. This result is at variance with the observation that our solution structure, like that of SCM, corresponds to a monomeric state of the protein, as demonstrated by the insensitivity of HSQC spectra to extreme dilution (down to 20 microM). On the basis of the solution structure of MNEI it is possible to propose that the main glucophores are hosted on loop L34, whereas the N-terminal and C-terminal regions host two other important interaction regions, centered around segments 6-9 and 94-96.

Amino Acid Sequence↗

PubMed1999

Solution structure of human beta-endorphin in helicogenic solvents: an NMR study.

Beta-endorphin is the largest natural opioid peptide. The knowledge of its bioactive conformation might be very important for the indirect mapping of the active site of opioid receptors. We have studied beta-endorphin in a variety of solution conditions with the goal of testing the intrinsic tendency of its sequence to assume a regular fold. We ran NMR experiments in water, dimethylsulfoxide and aqueous mixtures of methanol, ethylene glycol, trifluoroethanol, hexafluoracetone trihydrate and dimethylsulfoxide. The solvent in which the peptide is more ordered is the hexafluoracetone trihydrate/water mixture. The helical structure detected for beta-endorphin in this mixture at 300 K extends for the greater part of its address domain, hinting at a possible mechanism of interaction with opioid receptors: a two-point attachment involving an interaction of the helical part of the address domain (PLVTLFKNAIIKNAY) with one of the transmembrane helices and a classical interaction of the message domain (YGGF) with the receptor subsite common to all opioid receptors.

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

Solution conformational analysis of sodium complexed [Gly6]- and [Gly9]-antamanide analogs.

To investigate the conformational flexibility of metal-complexed cyclodecapeptides, we synthesized and studied two antamanide analogs, in which the phenylalanine residue in position 6 or 9 of the sequence was substituted by Gly. Previous conformational studies on antamanide suggested that these backbone regions are affected by conformational variation. The NMR conformational study showed a high degree of flexibility for the two analogs. With sodium ions, on the other hand, [Gly9]-antamanide was able to form a fairly stable equimolar complex, whereas [Gly6]-antamanide showed a conformational heterogeneity, with one prevailing conformer. For the [Gly9]-antamanide analog, the whole NMR data, combined with extensive theoretical calculations, were consistent with the presence of 1) two beta-turns of type I, centered on Gly9-Phe10 and Ala4-Phe5, respectively; 2) a central cavity with a six-carbonyl oxygen cage, optimal for a Na+ hexacoordination; 3) strongly H-bonded amide protons for residues 1 and 6, both involved in the formation of the two type I beta-turns, which, however, exhibited some fluctuations during the molecular dynamics simulations. For the [Gly6]-antamanide-Na+ complex the prevailing conformer was consistent with a more open structure, with the partial solvent exposure of all the amide protons; that is, the Gly residue in position 6 increases the flexibility of this critical site more than does the Gly in position 9. These data in some way parallel the results of the cytotoxicity tests on B16-F10 transformed cells for the two analogs: [Gly9]-antamanide is cytotoxic after 48 h exposure, whereas [Gly6]-antamanide is almost inactive. On the contrary, both analogs are practically inactive in vivo against phalloidin.

Amino Acid Substitution↗

PubMed1997

Conformational analysis of three NK1 tripeptide antagonists: a proton nuclear magnetic resonance study.

Two new peptides, tailored after Ac-Thr-D-Trp(CHO)-Phe-NMeBzl (TRI), namely, Ac-Thr-D-Trp(CHO)-Phe-NMe alpha MeBzl (TRA) and Ac-Thr-D-Trp(CHO)-Oic-NMeBzl (TOI), in which Phe is replaced by (3aS, 7aS)-octahydroindole-2-carboxylic acid, proved more potent and selective NK1 antagonists. The conformational properties of all three compounds were investigated in solution by NMR spectroscopy and those of TRI analyzed in greater detail by means of systematic computer-assisted modeling. All conformers whose energy differs by less than 9 kcal/mol from the absolute minimum are different from the conformer proposed in a previous molecular modeling study by the discovers of TRI. Parallel calculations for TRA and TOI yield low-energy conformers similar to those of TRI but in a slightly different order. Comparison of the shapes of low-energy conformers of all three peptides with those of four typical rigid NK1 antagonists shows that putative bioactive conformations are indeed present in solution.

Animals↗

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↗

PubMed1995

Structure-activity relationship of the leucine-based sorting motifs in the cytosolic tail of the major histocompatibility complex-associated invariant chain.

The cytosolic tail of the major histocompatibility complex-associated invariant chain protein contains two Leu-based motifs that both mediate efficient sorting to the endocytic pathway. Nuclear magnetic resonance data on a peptide of 27 residues corresponding to the cytosolic tail of human invariant chain indicate that in water at pH 7.4 the membrane distal motif Leu7-Ile8 lies within a nascent helix, while the membrane proximal motif Met16-Leu17 is part of a turn. The presence of a small amount of methanol stabilizes an alpha helix from Gln4 to Leu17 with a kink on Pro15. Point mutations of the cytosolic tail of the protein suggest that amino-terminal residues located in spatial proximity to the Leu motifs contribute to efficient internalization and targeting to endosomes in transfected COS cells. Residues on the spatially opposite side of the Leu motifs were, on the other hand, mutated with no measurable effect on targeting. Structural and biological data thus suggest that the signals are not continuous but consist of "signal patches" formed by the three-dimensional structure of the cytosolic tail of invariant chain.

Amino Acid Sequence↗

PubMed1994

Solution conformation of c-[Gln-Trp-Phe-Gly-Leu-Met], a NK-2 tachykinin antagonist.

The conformation of cyclo-[Gln-Trp-Phe-Gly-Leu-Met], a potent tachykinin antagonist selective for the NK-2 receptor, has been studied by 1H NMR spectroscopy in DMSO-d6 and in a DMSO-d6/H2O cryoprotective mixture in the temperature range 280-320 K. The NMR data cannot be interpreted on the basis of a single ordered conformation. An exhaustive search, based mainly on missing NOEs among skeleton protons, yields a description of the conformational state in solution consisting of a few interconverting structures that can explain all observed NMR parameters. The relative position of the side chains of key residues may be interpreted in terms of bioactive conformations.

Amino Acid Sequence↗

PubMed1993

Synthesis, biological activity, and conformational analysis of [pGlu6,N-MePhe8,Aib9] substance P (6-11): a selective agonist for the NK-3 receptor.

A highly potent and selective agonist to the tachykinin NK-3 receptor, [pGlu6,N-MePhe8,Aib9] substance P (6-11) (I), was synthesized via the solid phase method. The ED50 of I was 4 nM in the guinea pig ileum in the absence of atropine (NK-1+NK-3 receptors) and this agonist was 5000-fold less potent in the presence of atropine (NK-1 receptor). The analogue was virtually inactive in the rat vas deferens (NK-2 receptor). A detailed analysis of the solution conformation of this analogue in DMSO-d6 and in a DMSO-d6/H2O cryomixture was carried out by a combination of 1H-nmr 2D techniques (DQF-COSY, TOCSY, NOESY and ROESY) and model building based on empirical energy calculations. Peptide I exists as a mixture of isomers containing cis and trans Phe-N-MePhe peptide bonds. The main isomer, containing a cis Phe-N-MePhe peptide bond, shows a preferred folded conformation characterized by a type VI beta-turn with Phe and N-MePhe in the i + 1 and i + 2 positions. The turn is followed by a helical segment extending to the C-terminal. This conformation is compared to previously reported conformations of other selective tachykinin agonists and may be a promising lead for the design of novel NK-3 agonists with additional conformational constraints.

Amino Acid Sequence↗

PubMed1992

Structural determination of the active site of a sweet protein. A 1H NMR investigation of pMNEI.

pMNEI, a single chain sweet protein related to monellin, has been studied by means of 1H NMR at 500 MHz. A partial sequential assignment performed by means of the MCD method allowed the determination of the secondary structure of a large portion of the beta-sheet of pMNEI that contains a likely 'sweet finger': the loop connecting the beta-strands from residue 59 to residue 78, corresponding to segment 16-35 of the A chain of monellin. The detailed three-dimensional structure of the loop (Tyr66-Ala67-Ser68-Asp69), determined from several interresidue and intraresidue NOEs and subsequent energy minimization, shows that the side chains of Tyr66 and Asp69 fit our model of the sweet receptor in a manner very similar to that of the side chains of Phe and Asp of aspartame.

Amino Acid Sequence↗

PubMed1992

Conformational analysis of an opioid peptide in solvent media that mimic cytoplasm viscosity.

Many neuropeptides exert their action between the presynaptic vesicles and postsynaptic transmembrane receptors, crossing different layers of specialized cytoplasm. Biomimetic media usually employed to study bioactive peptides do not reproduce the physico chemical environment of cytoplasm--in particular, the high viscosity of this biological fluid. Here we describe a conformational study of a delta-selective opioid peptide, deltorphin I, at variable temperatures in several biocompatible media characterized by varying values of viscosity and dielectric constant. It was found that only viscosity, among these parameters, induces ordered conformations; that is, it acts as a conformational sieve. This finding suggests that the high viscosity of the intersynaptic fluid contributes, in addition to the membrane catalysis proposed by Schwyzer, in overcoming the so-called entropic barrier to the transition state of peptide-receptor interaction by selecting ordered conformations prior to receptor interaction. The folded conformer found in the 80:20 (v:v) DMSOd6/H2O cryoprotective mixture at 265 K has a shape consistent with those of rigid nonpeptidic opiates.

Amino Acid Sequence↗

PubMed1991

Conformation-activity relationship of tachykinin neurokinin A (4-10) and of some [Xaa8] analogues.

NKA (4-10), the C-terminal heptapeptide fragment (Asp-Ser-Phe-Val-Gly-Leu-Met-NH2) of tachykinin NKA, is more active than the parent native compound in the interaction with the NK-2 receptor. Substitution of Gly8 with the more flexible residue beta-Ala8 increases its selectivity with respect to other two known receptors (NK-1 and NK-3), whereas substitution with either D-Ala8 or GABA8 deprives the peptide of its biological activity. These findings can be interpreted by a conformational analysis based on NMR studies in DMSO-d6 and in a DMSO-d6/H2O cryoprotective mixture combined with internal energy calculations. NKA(4-10) is characterized by a structure containing a type I beta-turn extending from Ser5 to Gly8, followed by a gamma-turn centered on Gly8, whereas for [beta-Ala8]NKA(4-10) is possible to suggest a type I beta-turn extending from Ser5 to beta-Ala8, followed by a C8 turn comprising beta-Ala8 and Leu9 and by another beta-turn extending from beta-Ala8 to the terminal NH2. The preferred conformation of [beta-Ala8]NKA(4-10) is not compatible with models for NK-1 and NK-3 agonists proposed on the basis of rigid peptide agonists [Levian-Teitelbaum et al. (1989) Biopolymers 28, 51-64; Sumner & Ferretti (1989) FEBS Lett. 253, 117-120]. The preferred solution conformation of [beta-Ala8]NKA(4-10) may thus be considered as a likely bioactive conformation for NK-2 selective peptides.

Amino Acid Sequence↗

PubMed1989

NMR studies of a series of dehydrodermorphins.

The third and fifth aromatic residues of dermorphin, a potent mu-opioid peptide, and of its N-terminal fragments, from the pentapeptide to the parent heptapeptide amide, have been systematically substituted with Z-dehydrophenylalanine (delta-Phe) and/or Phe to investigate the conformation-activity relationship. The characterization in DMSO-d6 at 500 MHz indicates that, in this solvent, all peptides adopt essentially random, extended conformations, as a consequence of the strong solvation. The chemical shift of the methyl group of D-Ala is influenced by the precise orientation of the side chain of the third residue in a fashion that can be correlated to the mu potency, consistently with our model of mu-receptor. However, the complexes of the pentapeptides with 18-crown-6-ether, when dissolved in chloroform, adopt ordered, folded conformations, a behavior that closely parallels the CD observations in methanol.

Magnetic Resonance Spectroscopy↗

PubMed1984

Distal coronary power injection during percutaneous transluminal coronary angioplasty.

A modification of the standard percutaneous transluminal coronary angioplasty (PTCA) procedure is described using a power injector instead of a hand-held syringe for distal coronary contrast injections. This system was used in 215 dilatations without complications related to the power injector. We found this system to simplify the PTCA procedure and allowed for improved distal visualization even with the steerable PTCA catheters containing an intraluminal guide wire.

Angioplasty, Balloon↗

PubMed1973