Biomedical subjects

Y Shai

Publications and source records attributed to Y Shai.

85 records · Page 5Linked to original sources

PubMed1992

Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes.

Dermaseptin, a 34 amino-acid residue antimicrobial polypeptide [Mor, A., Nguyen, V. H., Delfour, A., Migliore-Samour, D., & Nicolas, P. (1991) Biochemistry 30, 8824-8830] was synthesized and selectively labeled at its N-terminal amino acid with either 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD), rhodamine, or fluorescein. The fluorescent emission spectra of the NBD-labeled dermaseptin displayed a blue-shift upon binding to small unilamellar vesicles (SUV), reflecting the relocation of the fluorescent probe to an environment of increased apolarity. Titrations of solutions containing NBD-labeled dermaseptin with SUV composed of zwitterionic or acidic phospholipids were used to generate binding isotherms, from which were derived surface partition constants of (0.66 +/- 0.06) x 10(4) M-1 and (2.8 +/- 0.3) x 10(4) M-1, respectively. The shape of the binding isotherms, as well as fluorescence energy transfer measurements, suggests that some aggregation of membrane-bound peptide monomers occurs in acidic but not in zwitterionic vesicles. The preferential susceptibility of the peptide to proteolysis when bound to zwitterionic but not to acidic SUV suggests that these aggregates might then penetrate a relatively short distance into the hydrophobic region of the acidic membrane. Furthermore, the results provide good correlation between the peptide's strong binding and its ability to permeate membranes composed of acidic phospholipids, as revealed by a dissipation of diffusion potential and a release of entrapped calcein from SUV.

Amino Acid Sequence↗

PubMed1992

Interaction of D-amino acid incorporated analogues of pardaxin with membranes.

The influence of specific L- to D-amino acid substitutions on the interaction of pardaxin, a shark repellent neurotoxin polypeptide, with phospholipid vesicles and human erythrocytes is described. Twelve modified, truncated, or fluorescently labeled [with the fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD) at their N-terminal amino acid] analogues of pardaxin were synthesized by a solid-phase method. Fluorescence measurements were used to monitor the interaction of the analogues with membranes [Rapaport, D., & Shai, Y. (1991) J. Biol. Chem. 266, 23769-23775]. Upon titration of solutions containing the NBD-labeled peptides with small unilamellar vesicles, the fluorescent emission spectra of all NBD-labeled peptides displayed similar blue-shifts, in addition to enhanced intensities, upon relocation of the probe to the more apolar environment. Binding isotherms were constructed from which surface partition constants, in the range of 10(4) M-1, were derived. The existence of an aggregation process, suggested by the shape of the binding isotherms, could be associated only with those analogues in which the N-helix (residues 1-9) was not perturbed. The alpha-helical content of the analogues was estimated by circular dichroism (CD) spectroscopy, both before and after binding to vesicles at neutral pH. The ability of the peptides to dissipate a diffusion potential and to cause calcein release, as well as to lyse human erythrocytes, served to functionally characterize the peptides. The results support a two alpha-helix model, with a bend at position 13, as best describing pardaxin in its membrane-bound state.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1992

Membrane interactions of the sodium channel S4 segment and its fluorescently-labeled analogues.

A 24-amino acid peptide corresponding to the S4 segment of the sodium channel was synthesized. In order to perform fluorescence energy transfer measurements and to monitor the interaction of the peptide with lipid vesicles, the peptide was selectively labeled with fluorescence probes at either its N- or C-terminal amino acids. The fluorescent emission spectra of 7-nitrobenz-2-oxa-1,3-diazol-4- yl-(NBD-)labeled analogues displayed blue shifts upon binding to small unilamellar vesicles (SUV), reflecting the relocation of the fluorescent probe to an environment of increased apolarity. The results revealed that both the N- and C-terminus of the S4 segment are located within the lipid bilayer. Titration of solutions containing NBD-labeled peptides with SUV was used to generate binding isotherms, from which surface partition constants, in the range of 10(4) M-1, were derived. The shape of the binding isotherms as well as fluorescence energy transfer measurements suggest that aggregation of peptide monomers within the membrane readily occurs in acidic but not in zwitterionic vesicles. Furthermore, the results provide good correlation between the incidence of aggregation in PC/PS vesicles and the ability of the peptides to permeate the vesicle's membrane. However, a transmembrane diffusion potential had no detectable effect on the location of the peptide within the lipid bilayer or on its aggregation state.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Aggregation and organization of pardaxin in phospholipid membranes. A fluorescence energy transfer study.

Fluorescence resonance energy transfer has been used to study the aggregation and organization of pardaxin and its analogues within lipid membranes. Peptide molecules labeled with 5- (and 6-) carboxyfluorescein at their N-terminal amino acid served as donors in these energy transfer measurements, whereas peptides labeled with 5- (and 6-) carboxytetramethylrhodamine at either their N- or C-terminal amino acid, served as acceptors. The membrane-permeating activity of the native molecule was maintained in the labeled peptides. Upon aggregation of the labeled peptides, fluorescence energy transfer was detected as a quenching of the donor fluorescence (520 nm), as well as an enhancement of the acceptor fluorescence (575 nm). Correlation exists between self-aggregation of the different analogues within membranes and their poreforming abilities. A comparison of the degrees of fluorescence energy transfer from N1-donor-labeled pardaxin to N1-acceptor-labeled pardaxin with the transfer efficiency observed in the interaction between the same donor and C1-acceptor-labeled pardaxin suggests that aggregates are formed in an ordered manner, with a preferentially parallel orientation of monomers within the aggregate. The extent of hetero-oligomer formation, i.e. complexes composed of two different analogue species, revealed that complementary charges contribute to peptide-peptide recognition within the lipid bilayer. Taken together, these results provide further support for the barrel stave model, involving parallel organization of monomers within the aggregate, as a description of the pore formation mechanism of pardaxin and its analogues.

Amino Acid Sequence↗

PubMed1992

In vivo imaging of insulin receptors in monkeys using 18F-labeled insulin and positron emission tomography.

We previously described a prosthetic group methodology for incorporating 18F into peptides and showed that 18F-labeled insulin (18F-insulin) binds to insulin receptors on human cells (IM-9 lymphoblastoid cells) with affinity equal to that of native insulin (1). We now report studies using 18F-insulin with positron emission tomography to study binding to insulin receptors in vivo. Positron emission tomography scans were performed in six rhesus monkeys injected with 0.3-1.4 mCi of 18F-insulin (approximately 0.1 nmol, SA 4-11 Ci/mumol). Integrity of the tracer in blood, determined by immunoprecipitation, was 94% of control for the first 5 min and decreased to 31% by 30 min. Specific, saturable uptake of 18F was observed in the liver and kidney. Coinjection of unlabeled insulin (200 U, approximately 1 nmol) with the 18F-insulin reduced liver and increased kidney uptake of the labeled insulin. Liver radioactivity was decreased by administration of unlabeled insulin at 3 min, but not 5 min, after administration of the tracer, while some kidney radioactivity could be displaced 5 min after injection. Clearance of 18F was predominantly in bile and urine. 18F-insulin is a suitable analogue for studying insulin receptor-ligand interactions in vivo, especially in the liver and kidney.

Animals↗

PubMed1991

Interaction of fluorescently labeled pardaxin and its analogues with lipid bilayers.

Fluorescence measurements were used to monitor the interaction of the neurotoxin pardaxin and its analogues with membranes. Eight peptides were selectively labeled with the fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl, either at their N-terminal or at their C-terminal. No detectable changes in membrane permeability or hemolytic activity were observed upon modification. Upon the titration of solutions containing the different peptides with small unilamellar vesicles, the fluorescent emission spectra of 7-nitrobenz-2-oxa-1,3-diazole-4-yl-labeled pardaxin and its analogues, but not those of control peptides, displayed blue shifts in addition to enhanced intensities upon relocation of the probe to a more apolar environment. The results revealed that the N terminus of pardaxin is buried within the lipid bilayer while the C terminus is located at the bilayer's surface. Binding isotherms were obtained from the observed increases in the fluorescence emission yields, from which surface partition constants, in the range of 10(4) M-1, were in turn derived. The existence of an aggregation process was suggested by the shape of the binding isotherms. Furthermore, the results show good correlation between the incidence of aggregation and the ability of the different analogues to induce the release of relatively large molecules from vesicles. As such, our results suggest that the mechanism of pore formation employed by pardaxin and its analogues could be described by the "barrel stave" model.

4-Chloro-7-nitrobenzofurazan↗

PubMed1991

pH-dependent pore formation properties of pardaxin analogues.

The interaction of pardaxin, a shark-repellent neurotoxin, and its charge-modified analogues with vesicles and human erythrocytes is described. The following six analogues and derivatives were synthesized by a solid phase method: [Glu8, Glu16]pardaxin, [N1-succinamido,Glu8,Glu16]pardaxin, [N1,Lys8,Lys16-triacetyl]pardaxin, des-[1----9]pardaxin (Shai, Y., Bach, D., and Yanovsky, A. (1990) J. Biol. Chem. 265, 20202-20209), and des-[1----9] [Glu16]pardaxin. The relative hydrophobic characteristics of the analogues were examined using reverse-phase high performance liquid chromatography. The pH-dependent spectroscopic and functional characteristics of the analogues were also investigated at either neutral or acidic pH. Spectroscopic characterization was achieved by measuring circular dichroism both before and after binding to vesicles, at either neutral or acidic pH. The ability of the peptides to dissipate a diffusion potential, to cause calcein release or the pH-dependent release of 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt/p-xylene-bis[pyridinium bromide] from sonicated unilamellar liposomes, as well as measurements of cytolytic activity on human erythrocytes, served to functionally characterize the peptides. We show a direct correlation between alpha-helical content, the analogues' hydrophobicity, and their pore-forming properties at the different pH values tested. We also demonstrate that the charge of the N terminus and of the peptide backbone, but not of the C terminus, affects the secondary structure as well as the activities of the analogues. Finally, we show that the cytolytic activity of pardaxin at neutral pH is not retained by any of the analogues.

Amino Acid Sequence↗

PubMed1990

Channel formation properties of synthetic pardaxin and analogues.

Six analogues of teh 33-residue shark repellent neurotoxin pardaxin were synthesized by the solid phase method: [Ala13]pardaxin, [Gly14,Gly15]pardaxin, des[1----9]pardaxin, [N1-succinamido]pardaxin, C33-dihydroxyethylamido]pardaxin, and C33-[diaminoethylamido]pardaxin. The spectroscopic and functional characterizations of the analogues are described. The peptides were characterized spectroscopically by circular dichroism (CD) before and after binding to soybean vesicles. They were characterized functionally by measuring their potential to evoke the dissipation of diffusion potential and calcein release from sonicated unilamellar soybean liposomes, by determining their ability to create single channels in planar bilayers, and by measuring their cytolytic activity on human erythrocytes. The behavior of the analogues modified at the C terminus is similar to that of pardaxin. [N'-succinamido]Pardaxin, however, reveals an increase in alpha-helicity both alone and in the presence of liposomes. It has the same potency as pardaxin to dissipate diffusion potential, to evoke calcein release and to produce single channels in lipid bilayers, but at a slower rate than that of pardaxin. It has more than 70-fold less cytolytic activity than pardaxin. [Ala13] Pardaxin has twice the alpha-helical content than pardaxin, both alone and in the presence of vesicles, yet it has less effect on the diffusion potential and calcein release, and it does not have cytolytic activity on human erythrocytes. Both [Gly14,Gly15]pardaxin and des[1----9]pardaxin are much less potent than pardaxin in all effects. However des[1----9]pardaxin exhibits a slight change in alpha-helicity upon binding to vesicles, whereas [Gly14,Gly15]pardaxin does not. The results support a model in which pardaxin is composed of two putative alpha-helices separated by proline. The N-terminal alpha-helix is important for the insertion of the peptide to the lipid bilayer, and the C-terminal amphiphilic alpha-helix is the ion channel lining segment of pardaxin.

Amino Acid Sequence↗

PubMed1989

Antisense peptide recognition of sense peptides: sequence simplification and evaluation of forces underlying the interaction.

Structural principles were studied which underlie the recognition of sense peptides (sense DNA encoded) by synthetic peptides encoded in the corresponding antisense strand of DNA. The direct-readout antisense peptides corresponding to ribonuclease S-peptide bind to an affinity matrix containing immobilized S-peptide with significant selectivity and with dissociation constants in the range of 10(-6) M as judged by analytical affinity chromatography. Synthetic, sequence-modified forms of antisense peptides also exhibit substantial binding affinity, including a "scrambled" peptide in which the order of residue positions is changed while the overall residue composition is retained. The antisense mutants, as the original antisense peptides, bind at saturation with greater than 1:1 stoichiometry to immobilized S-peptide. The data suggest significant sequence degeneracy in the interaction of antisense with sense peptide. In contrast, selectivity was confirmed by the inability of several control peptides to bind to immobilized S-peptide. The idea was tested that the hydropathic pattern of the amino acid sequence serves to induce antisense peptide recognition. A hydropathically sequence-simplified mutant of antisense peptide was made in which all strongly hydrophilic (charged) residues were replaced by Lys, all strongly hydrophobic residues by Leu, and all weakly hydrophilic and hydrophobic residues by Ala, except Gly which was unchanged. This "KLAG" mutant also binds to immobilized S-peptide, with an affinity only an order of magnitude less than that with the original antisense peptide and with multiple stoichiometry. Mutants of the KLAG model, in which the hydropathic pattern was changed substantially, exhibited a lower binding affinity for S-peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1989

18F-labeled insulin: a prosthetic group methodology for incorporation of a positron emitter into peptides and proteins.

In the present study we synthesize 18F-labeled insulin of high specific radioactivity. A new prosthetic group methodology, in which [18F]fluoride displaces a bromide group of 4-(bromomethyl)-benzoylamine intermediates, was used. The 4-(fluoromethyl)benzoyl product was chemically stable. 18F-Labeled insulin retains the essential biological properties of native insulin, as measured in vitro by binding to insulin receptors on human cells and stimulation of glucose metabolism in rat adipocytes. The overall process can be carried out speedily to yield a product of sufficient purity to permit in vivo studies. The method appears to be applicable to a wide variety of peptides.

Adipose Tissue↗

PubMed1988

Sequencing and synthesis of pardaxin, a polypeptide from the Red Sea Moses sole with ionophore activity.

Pardaxin, an amphipathic polypeptide secreted by the Red Sea flatfish Pardachirus marmoratus whose sequence is NH2-G-F-F-A-L-I-P-K-I-I-S-S-P-L-F-K-T-L-L-S-A-V-G-S-A-L-S-S-S-G-G-Q-E, was synthesized by the solid-phase method. The structure was verified by sequencing. The synthetic polypeptide changed the resistance of lipid bilayers by forming pores. At 10(-7)-10(-8) M, the synthetic pardaxin increased the frequency of the spontaneous release of quanta of acetylcholine at the neuromuscular junction by up to 100-fold, resembling the native product. Synthetic pardaxin seems to be a suitable tool for investigating the molecular structures underlying channel selectivity.

Acetylcholine↗

PubMed1987

Anti-sense peptide recognition of sense peptides: direct quantitative characterization with the ribonuclease S-peptide system using analytical high-performance affinity chromatography.

The ability of peptides coded by the anti-sense strand of DNA to interact specifically with peptides coded by the sense strand has been evaluated. The sense peptide examined, ribonuclease S-peptide, was immobilized on a coated silica affinity chromatographic matrix. Anti-sense peptides were synthesized on the basis of the anti-sense DNA sequence for the S-peptide region in native pancreatic ribonuclease A. The interaction of synthetic anti-sense peptides with sense peptide was quantitated from the degree of retardation during chromatographic elution on the sense peptide affinity matrix in buffers with and without soluble competing sense peptide. Sense/anti-sense peptide interactions were found to occur with significant affinities with each of two anti-sense 20-residue peptides of opposite amino-to-carboxyl orientations and to weaken progressively with decreasing length of anti-sense peptide. The substantial chromatographic retardation of anti-sense peptides was specific, since it decreased as expected with increasing concentration of the soluble competing S-peptide, could not be mimicked by the elution of several control peptides (including S-peptide itself) on the S-peptide matrix, and did not occur with a blank chromatographic matrix (no S-peptide attached). The stoichiometry of anti-sense peptide binding to immobilized sense peptide was found to be far greater than 1:1, and at least 4-5:1, for the two 20-mer anti-sense peptides. In sum, the analytical affinity chromatographic experiments have established quantitatively that anti-sense peptide binding to sense peptides occurs in the ribonuclease S-peptide case and have identified some structural elements that govern these interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMedDate not supplied

What studies of fusion peptides tell us about viral envelope glycoprotein-mediated membrane fusion (review).

This review describes the numerous and innovative methods used to study the structure and function of viral fusion peptides. The systems studied include both intact fusion proteins and synthetic peptides interacting with model membranes. The strategies and methods include dissecting the fusion process into intermediate stages, comparing the effects of sequence mutations, electrophysiological patch clamp methods, hydrophobic photolabelling, video microscopy of the redistribution of both aqueous and lipophilic fluorescent probes between cells, standard optical spectroscopy of peptides in solution (circular dichroism and fluorescence) and attenuated total reflection-Fourier transform infrared spectroscopy of peptides bound to planar bilayers. Although the goal of a detailed picture of the fusion pore has not been achieved for any of the intermediate stages, important properties useful for constraining the development of models are emerging. For example, the presence of alpha-helical structure in at least part of the fusion peptide is strongly correlated with activity; whereas, beta-structure tends to be less prevalent, associated with non-native experimental conditions, and more related to vesicle aggregation than fusion. The specific angle of insertion of the peptides into the membrane plane is also found to be an important characteristic for the fusion process. A shallow penetration, extending only to the central aliphatic core region, is likely responsible for the destabilization of the lipids required for coalescence of the apposing membranes and fusion. The functional role of the fusion peptides (which tend to be either nonpolar or aliphatic) is then to bind to and dehydrate the outer bilayers at a localized site; and thus reduce the energy barrier for the formation of highly curved, lipidic 'stalk' intermediates. In addition, the importance of the formation of specific, 'higher-order' fusion peptide complexes has also been shown. Recent crystallographic structures of core domains of two more fusion proteins (in addition to influenza haemagglutinin) has greatly facilitated the development of prototypic models of the fusion site. This latter effort will undoubtedly benefit from the insights and constraints gained from the studies of fusion peptides.

Amino Acid Sequence↗