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F Formaggio

Publications and source records attributed to F Formaggio.

At least 19 recordsLinked to original sources
PubMed2006

Location and aggregation of the spin-labeled peptide trichogin GA IV in a phospholipid membrane as revealed by pulsed EPR.

The lipopeptaibol trichogin GA IV is a 10 amino acid-long residue and alpha-aminoisobutyric acid-rich antibiotic peptide of fungal origin. TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) spin-labeled analogs of this membrane active peptide were investigated in hydrated bilayers of dipalmitoylphosphatidylcholine by electron spin echo envelope modulation (ESEEM) spectroscopy and pulsed electron-electron double resonance (PELDOR). Since, the ESEEM of the spin label appears to be strongly dependent on the presence of water molecules penetrated into the membrane, this phenomenon was used to study the location of this peptide in the membrane. This was achieved by comparing the ESEEM spectra for peptides labeled at different positions along the amino acid sequence with spectra known for lipids with spin labels at different positions along the hydrocarbon chain. To increase the ESEEM amplitude and to distinguish the hydrogen nuclei of water from lipid protons, membranes were hydrated with deuterated water. The PELDOR spectroscopy technique was chosen to study peptide aggregation and to determine the mutual distance distribution of the spin-labeled peptides in the membrane. The location of the peptide in the membrane and its aggregation state were found to be dependent on the peptide concentration. At a low peptide/lipid molar ratio (less than 1:100) the nonaggregated peptide chain of the trichogin molecules lie parallel to the membrane surface, with TOAC at the 4th residue located near the 9th-11th carbon positions of the sn-2 lipid chain. Increasing this ratio up to 1:20 leads to a change in peptide orientation, with the N-terminus of the peptide buried deeper into membrane. Under these conditions peptide aggregates are formed with a mean aggregate number of about N = 2. The aggregates are further characterized by a broad range of intermolecular distances (1.5-4 nm) between the labels at the N-terminal residues. The major population exhibits a distance of approximately 2.5 nm, which is of the same order as the length of the helical peptide. We suggest that the constituting monomers of the dimer are antiparallel oriented.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2005

On the orange color of Z-Trp-ONPo.

Out of all nitrophenyl esters of N(alpha)-protected alpha-amino acids Z-Trp-ONP(o) is the only one which is deep orange colored in the crystalline state. Any change in N(alpha)-protection, nature of amino acid, spatial separation between Trp and the ester-group or position of the nitro-substitutent in the aromatic ring of the ester function results in a loss of this characteristic property. We solved the molecular and crystal structure of Z-l-Trp-ONP(o) by X-ray diffraction analysis and investigated its color changes and visible (vis) and infrared (IR) absorption spectra in the solid state as a function of the amino acid derivative/KBr (w/w) ratio in the pellets. This investigation was extended to toluene solutions of different Z-Trp-ONP(o) concentrations by use of vis absorption and proton magnetic resonance spectroscopic techniques. The onset of the orange color correlates closely with the appearance of a concentration-dependent absorption band near 500 nm and concentration-dependent shifts of the urethane and indole NH proton resonances. Our observations can be explained by the formation of an intermolecular charge transfer complex involving the Trp indole and the -ONP(o) nitrophenyl as the donor and the acceptor moieties, respectively.

Color↗
PubMed2005

A topographically and conformationally constrained, spin-labeled, alpha-amino acid: crystallographic characterization in peptides.

2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) is a topographically and conformationally restricted, nitroxide containing, C(alpha)-tetrasubstituted alpha-amino acid. Here, we describe the molecular and crystal structures, as determined by X-ray diffraction analyses, of a TOAC terminally protected derivative, the cyclic dipeptide c(TOAC)(2).1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) solvate, and five TOAC-containing, terminally protected, linear peptides ranging in length from tetra- to hepta-peptides. Incipient and fully developed, regular or distorted 3(10)-helical structures are formed by the linear peptides. A detailed discussion on the average geometry and preferred conformation for the TOAC piperidine ring is also reported. The X-ray diffraction structure of an intramolecularly cyclized side product resulting from a C-activated TOAC residue has also been determined.

Crystallography, X-Ray↗
PubMed2004

New tools for the control of peptide conformation: the helicogenic Calpha-methyl, Calpha-cyclohexylglycine.

The novel Calpha-tetrasubstituted alpha-amino acid Calpha-methyl, Calpha-cyclohexylglycine was prepared by hydrogenation of its Calpha-methyl, Calpha-phenylglycine precursor. Terminally protected homodi-, homotri-, and homotetrapeptides from Calpha-methyl, Calpha-cyclohexylglycine and co-oligopeptides to the pentamer level in combination with Gly or alpha-aminoisobutyric acid residues were prepared by solution methods and fully characterized. The results of a conformational analysis, performed by use of Fourier transform infrared (FT-IR) spectrophotometer absorption, 1H NMR, and X-ray diffraction techniques, support the contention that this Calpha-methylated, Cbeta-trisubstituted aliphatic alpha-amino acid is an effective beta-turn and 3(10)-helix inducer in tri- and longer peptides as its Calpha-methyl valine parent compound, but partially divergent from the corresponding aromatic Calpha-methyl, Calpha-diphenylmethylglycine residue, known to promote folded and fully extended structures to a significant extent in these oligomers.

Alanine↗
PubMed2003

Design and function of a conformationally restricted analog of the influenza virus fusion peptide.

A conformationally restricted analog of the N-terminal 12-residue peptide segment of the HA2 subunit of the PPV/34, PR/8/34, and Jap/57 strains of influenza virus hemagglutinin was synthesized containing three residues of Calpha-methyl-valine. This peptide has a higher content of helical structure in the presence of 50% trifluoroethanol or when interacting with liposomes of egg phosphatidylcholine compared with the conformationally more flexible control peptide with the native sequence. The control and analog peptides had opposite effects on membrane curvature as measured by shifts in the bilayer-to-hexagonal phase transition temperature of a synthetic phosphatidylethanolamine derivative. The control peptide promoted more negative curvature, particularly at acidic pH and was also more potent than the analog in promoting lipid mixing. The results indicate that the ability of the peptide to sample a broader range of conformations is required for the influenza fusion peptide to destabilize membranes and that a rigid helical structure is less fusogenic. The difference between the two peptides and between pH 7.4 and pH 5.0 show a correlation between the ability to promote negative curvature and to accelerate lipid mixing.

Calorimetry, Differential Scanning↗
PubMed2002

Structural features and conformational equilibria of 310-helical peptides in solution by spectroscopic and molecular mechanics studies.

The structural features and conformational equilibria of a series of short, linear Calpha-methylvaline [(alphaMe)Val]-based peptides in methanol were investigated by combining fluorescence resonance energy transfer measurements and molecular mechanics data. IR spectra were employed to determine their secondary structure, which exhibits an intramolecularly H-bonded, 3(10)-helix conformation that is affected by backbone distortions that are enhanced by the shortness of the main chain.

Models, Chemical↗
PubMed2001

Intramolecular, intermolecular, and heterogeneous nonadiabatic dissociative electron transfer to peresters.

The electron transfer to peresters was studied by electrochemical means in N,N-dimethylformamide. The reduction was carried out by three independent methods: (i) heterogeneously, by using glassy carbon electrodes, (ii) homogeneously, by using electrogenerated radical anions as the donors, and (iii) intramolecularly, by using purposely synthesized donor-spacer-acceptor (D-Sp-A) systems. Convolution analysis of the heterogeneous data led to results in excellent agreement with the dissociative electron transfer theory. The homogeneous redox catalysis data also confirmed the reduction mechanism. The cyclic voltammetries of the D-Sp-A molecules could be simulated, leading to determination of the corresponding intramolecular dissociative rate constants. Analysis of the results showed that, regardless of the way by which the acceptor is reduced, the investigated dissociative electron transfers are strongly nonadiabatic and, particularly, that the experimental rates are several orders of magnitude smaller than the adiabatic limit. A possible mechanism responsible for the observed behavior is discussed.

Journal Article↗
PubMed2001

Solution structure, dimerization, and dynamics of a lipophilic alpha/3(10)-helical, C alpha-methylated peptide. Implications for folding of membrane proteins.

The solution structure and the dimerization behavior of the lipophilic, highly C(alpha)-methylated model peptide, mBrBz-Iva(1)-Val(2)-Iva(3)-(alphaMe)Val(4)-(alphaMe)Phe(5)-(alphaMe)Val(6)-Iva(7)-NHMe, was studied by NMR spectroscopy and molecular dynamics simulations. The conformational analysis resulted in a right-handed 3(10)/alpha-helical equilibrium fast on the NMR time scale with a slight preference for the alpha-helical conformation. The NOESY spectrum showed intermolecular NOEs due to an aggregation of the heptapeptide. In addition, temperature-dependent diffusion measurements were performed to calculate the hydrodynamic radius. All these findings are consistent with an antiparallel side-by-side dimerization. The structure of the dimeric peptide was calculated with a simulated annealing strategy. The lipophilic dimer is held together by favorable van der Waals interactions in the sense of a bulge fitting into a groove. The flexibility of the helical conformations concerning an alpha/3(10)-helical equilibrium is shown in a 3 ns molecular dynamics simulation of the resulting dimeric structure. Both overall helical structures of each monomer and the antiparallel mode of dimerization are stable. However, transitions were seen of several residues from a 3(10)-helical into an alpha-helical conformation and vice versa. Hence, this peptide represents a good model in which two often-discussed aspects of hierarchical transmembrane protein folding are present: i <-- i + 3 and i <-- i + 4 local H-bonding interactions cause a specific molecular shape which is then recognized as attractive by other surrounding structures.

Dimerization↗
PubMed2001

The secondary structure of a membrane-modifying peptide in a supramolecular assembly studied by PELDOR and CW-ESR spectroscopies.

The new technique of pulsed electron-electron double resonance in electron spin-echo (PELDOR) in combination with the CW-ESR method has been used to investigate the secondary structure of a double spin-labeled peptide (the [TOAC-1,8]-analogue of the peptaibol antibiotic trichogin GA IV) that is hidden into a tetrameric supramolecular assembly of unlabeled peptide molecules. The magnetic dipole-dipole relaxation of spin labels has been experimentally studied in glassy solutions of the double-labeled peptide frozen to 77 K in a mixture of chloroform-toluene with an excess of unlabeled peptide. The PELDOR signal oscillations have been observed at high degrees of dilution with unlabeled peptide. The intramolecular distance between the spin labels of the peptide molecule in the aggregate has been determined from the oscillation frequency to be 15.7 A which is close to the value of (approximately equal to) 14 A calculated for a 3(10)-helical structure. Estimation of the fraction of this ordered secondary structure shows that about 19% of the peptide molecules in aggregates are folded in the 3(10)-helical conformation. The present experimental results are consistent with our molecular model presented in J. Am. Chem. Soc. 2000, 122, 3843-3848, wherein four amphiphilic 3(10)-helical peptide molecules form a vesicular system with the polar amino acid side chains pointing to the interior, and the apolar side chains, to the exterior of the cluster. The experimental data were compared with the results obtained with other techniques.

Journal Article↗
PubMed2001

Agonist activity at the kinin B1 receptor: structural requirements of the central tetrapeptide.

A series of analogues of desArg(9)-Lys-bradykinin (BK), Lys-Arg-X-Ac(n)c-X-Ser-Pro-Phe, in which the spacer X-Ac(n)c-X replaces the central tetrapeptide Pro-Pro-Gly-Phe of BK, have been synthesized and functionally characterized at the B1 receptor. The 1-aminocycloalkane-1-carboxylic acids (Ac(6)c, Ac(7)c, Ac(8)c, Ac(9)c, Ac(12)c) were incorporated to impart conformational constraint and probe the importance of the hydrophobicity of the residue in the central position. The linker is varied in length (X = Gly, betaAla, gammaAbu) to examine the optimal distance between the biologically important residues at the N- and C-termini. The biological assays indicate that the optimal length is obtained with X = Gly, with reduced activities for the longer linkers. Although the size of the central cyclic amino acid does not significantly alter the biological activity, the hydrophobic residue Ac(n)c which may tether the peptide in the membrane environment is required (Lys-Arg-Gly-Gly-Gly-Ser-Pro-Phe is inactive). Two of the analogues, Lys-Arg-Gly-Ac(7)c-Gly-Ser-Pro-Phe and Lys-Arg-gammaAbu-Ac(7)c-gammaAbu-Ser-Pro-Phe, have been structurally characterized in the presence of a zwitterionic lipid environment by high-resolution NMR. Both compounds have similar structural features, differing greatest in the distance between the termini (9 and 15 A for the Gly- and gammaAbu-containing analogues, respectively). The correlation of the smaller distance with activity at the B1 receptor is in complete accord with the results from our previous examination of Lys-Arg-NH-(CH(2))(11)-CO-Ser-Pro-Phe. With the results from this series of compounds we are beginning to define some of the molecular descriptors important for activity at the B1 BK receptor.

Animals↗
PubMed2001

Control of peptide conformation by the Thorpe-Ingold effect (C alpha-tetrasubstitution).

The preferred conformations of peptides heavily based on the currently extensively exploited achiral and chiral alpha-amino acids with a quaternary alpha-carbon atom, as determined by conformational energy computations, crystal-state (x-ray diffraction) analyses, and solution ((1)H-NMR and spectroscopic) investigations, are reviewed. It is concluded that 3(10)/alpha-helical structures and the fully extended (C(5)) conformation are preferentially adopted by peptide sequences characterized by this family of amino acids, depending upon overall bulkiness and nature (e.g., whether acyclic or C(alpha) (i) <--> C(alpha) (i) cyclized) of their side chains. The intriguing relationship between alpha-carbon chirality and bend/helix handedness is also illustrated. gamma-Bends and semiextended conformations are rarely observed. Formation of beta-sheet structures is prevented.

Amino Acids↗
PubMed2001

Lipopeptaibols, a novel family of membrane active, antimicrobial peptides.

Lipopeptaibols are members of a novel group of naturally occurring, short peptides with antimicrobial activity, characterized by a lipophilic acyl chain at the N-terminus, a high content of the turn/helix forming alpha-aminoisobutyric acid and a 1,2-amino alcohol at the C-terminus. The amino acid sequences range from 6 to 10 residues and the fatty acyl moieties from 8 to 15 carbon atoms. The peptide portion of lipopeptaibols can be shorter than those of the nonlipidated peptaibols that range from 10 to 19 amino acid residues. The longest peptides fold into a mixed 3(10)/alpha helix, whereas the shortest peptides tend to adopt a beta-turn/sheet structure. Using solution methodologies, a series of analogues of trichogin GA IV was synthesized which allowed determination of the minimal lipid chain and peptide main-chain lengths for the onset of membrane activity and exploitation of a number of spectroscopic techniques aimed at determining its preferred conformation under a variety of conditions and investigating in detail its mode of interaction with, and its effect on, the phospholipid membranes.

Amino Acid Sequence↗
PubMed2001

Partial [alphaMe]Aun scan of [l-Leu11-OMe]-trichogin GA IV, a membrane active synthetic precursor of the natural lipopeptaibol.

We synthesized using solution-phase methods three analogs of [l-Leu11-OMe] trichogin GA IV, a membrane active synthetic precursor of the lipopeptaibol antibiotic in which the N-terminal n-octanoyl group and each of the three Aib residues in positions 1, 4 and 8 are replaced by an acetyl group and the lipophilic Calpha,alpha-disubstituted glycine l-(alphaMe)Aun, respectively [partial (alphaMe)Aun scan]. FT-IR absorption and CD analyses unequivocally show that the main three-dimensional structural features of [l-Leu11-OMe] trichogin GA IV are preserved in the analogs. Also, [l-Leu11-OMe] trichogin GA IV and the three Nalpha-acetylated l-(alphaMe)Aun analogs exhibit strictly comparable membrane-modifying properties. Taken together, these results strongly favor the conclusion that a shift of the long hydrocarbon moiety from the Nalpha-blocking group to the side-chain of the 1, 4 or 8 residue does not have any significant effect on the conformational properties or the membrane activity of [l-Leu11-OMe] trichogin GA IV and, by extension, of the natural lipopeptaibol.

Anti-Bacterial Agents↗
PubMed2001

Solid-phase synthesis of peptides containing the spin-labeled 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC).

2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino4-carboxylic acid (TOAC) is a nitroxide spin-labeled, achiral Calpha-tetrasubstituted amino acid recently shown to be not only an effective beta-turn and 3(10)/alpha-helix promoter in peptides, but also an excellent rigid electron paramagnetic resonance probe and fluorescence quencher. Here, we demonstrate that TOAC can be effectively incorporated into internal positions of peptide sequences using Fmoc chemistry and solid-phase synthesis in an automated apparatus.

Amino Acids↗
PubMed2001

Ac10c: a medium-ring, cycloaliphatic Calpha,alpha-disubstituted glycine. Incorporation into model peptides and preferred conformation.

Two complete series of N-protected oligopeptide esters to the pentamer level from 1-amino-cyclodecane-1-carboxylic acid (Ac10c), an alpha-amino acid conformationally constrained through a medium-ring Calphai <--> Calphai cyclization, and either the L-Ala or Aib residue, along with the N-protected Ac10c monomer and homo-dimer alkylamides, were synthesized using solution methods and fully characterized. The preferred conformation of these model peptides was assessed in deuterochloroform solution using FT-IR absorption and 1H NMR techniques. Furthermore, the molecular structures of two derivatives (Z-Ac10c-OH and Fmoc-Ac10c-OH) and two peptides (the dipeptide ester Z-Ac10c-L-Phe-OMe and the tripeptide ester Z-Aib-Ac10c-Aib-OtBu) were determined in the crystal state using X-ray diffraction. The experimental results support the view that beta-bends and 3(10)-helices are preferentially adopted by peptides rich in Ac10c, the third largest cycloaliphatic C(alpha,alpha)-disubstituted glycine known. This investigation allowed us to complete 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 our recent proposal of the 'Ac(n)c scan' concept.

Crystallography, X-Ray↗
PubMed2001

Analogs of the antimicrobial peptide trichogin having opposite membrane properties.

Four analogs of the antimicrobial peptide trichogin GA IV were studied. Their sequences are as follows: GT, n-octanoyl-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe; ST, n-octanoyl-Aib-Ser-Leu-Aib-Ser-Ser-Leu-Aib-Ser-Ile-Leu-OMe; BT, n-octanoyl-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-OMe; and DT, n-octanoyl-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-OMe. The trichogin GA IV differs from GT only in the nature of the C-terminal residue, being a 1,2 aminoalcohol (leucinol) in the case of the parent peptide. Compared with GT, ST has an increased amphiphilicity. In contrast, BT has little amphiphilicity being composed only of hydrophobic amino acids. DT is an octanoylated head-to-tail dimer of BT. We show that BT and DT lower the bilayer-to-hexagonal phase transition temperature (T(H)) of dipalmitoleoylphosphatidylethanolamine, indicating that the peptides promote negative curvature. These two peptides, composed of only hydrophobic amino acids, have their bulkier groups on one face of the helix, suggesting that they may penetrate membranes at an oblique angle. In contrast, GT and ST, like trichogin itself, increase TH, promoting positive curvature. These peptides have contrasting membrane lytic activities. Whereas DT and BT did not produce leakage of aqueous contents, GT and ST, like trichogin, did cause rapid leakage. The leakage activity with liposomes also correlates with the greater potency of GT and ST, compared with the hydrophobic analogs, in their hemolytic and bacteriostatic action. ST has greater lytic ability than GT in liposomal leakage as well as hemolysis. We also measured the rate of peptide-promoted lipid mixing as an indication of membrane fusion. BT produced lipid mixing only with large unilamellar vesicles enriched with dioleoylphosphatidylethanolamine; ST did not produce lipid mixing, as its apparent reduction of energy transfer proved to be artifactual. Quasi-elastic light scattering of large unilamellar vesicles was also carried out after adding ST and BT. Peptide BT, but not ST, was able to aggregate large unilamellar vesicles. Thus, one of the properties of BT that leads to the induction of lipid mixing is that it is able to aggregate vesicles, placing the bilayers in juxtaposition. Thus, the two pairs of peptides, BT and DT vs GT and ST, exhibit contrasting behaviour with respect to a number of membrane biophysical properties. This occurs despite the fact that the chemical structures of the peptides are rather similar. Such distinct behavior is also reflected in their hemolytic and bacteriostatic actions.

Amino Acid Sequence↗
PubMed2000

The first water-soluble 3(10)-helical peptides.

Two water-soluble 3(10)-helical peptides are synthesized and fully characterized for the first time. The sequence of these terminally blocked heptamers comprises two residues of the Calpha-trisubstituted alpha-amino acid 2-amino-3-[1-(1,4,7-triazacyclononyl)]propanoic acid and five residues of a Calpha-tetrasubstituted alpha-amino acid (either alpha-aminoisobutyric acid or isovaline). Using CD and NMR techniques we were able to show that both heptapeptides are well structured in water, and that the type of conformation adopted is indeed the ternary 3(10)-helix.

Journal Article↗