Biomedical subjects

Y Shai

Publications and source records attributed to Y Shai.

At least 73 records · Page 4Linked to original sources

PubMed1995

Synthetic peptides corresponding to the four P regions of Electrophorus electricus Na+ channel: interaction with and organization in model phospholipid membranes.

The hydropathy plot of the alpha subunit of the voltage-gated Na+ channel reveals four homologous repeats, each of which is homologous to Shaker type K+ channel monomer and contains six putative transmembrane segments and a hydrophobic segment within the loop connecting transmembrane segments S5 and S6. Current models predict that the four homologous segments [designated H5 or P regions (PR)] from the S5-S6 loop of each repeat lie in the aqueous pore. Peptides corresponding to the P regions of the four domains of the Electrophorus electricus (eel) Na+ channel (25-36 aa long, designated as PR-I, PR-II, PR-III, and PR-IV) and a 23-mer preceding PR-II (designated pre-PR-II) were synthesized and fluorescently labeled. The segments were then structurally and functionally characterized for their interaction with phospholipid membranes. Although the sequences of the four P regions are significantly different, they all bind to zwitterionic phospholipid membranes with similar partition coefficients (approximately 10(4) M-1). The pre-PR-II does not bind membranes at all. Resonance energy transfer measurements, between donor/acceptor-labeled pairs of peptides, revealed that besides the PR-I/PR-III pair, all other pairs form heteroaggregates but do not coassemble with unrelated membrane-bound peptide. Circular dichroism (CD) spectroscopy revealed that PR-I, PR-II, and PR-III adopt similar partial alpha-helical structures (approximately 30%) in 40% trifluoroethanol and in solutions of 1% sodium dodecylsulfate (SDS). The PR-IV (36 aa) adopts approximately 18% alpha-helical structure, and pre-PR-II gives a low CD signal. These findings are in line with proposed models in which the P regions are packed in close proximity in the lumen of the hydrophobic core of the channel. Furthermore, the finding that the PRs adopt similar partial alpha-helical structures in two different hydrophobic environments might suggest that partial alpha-helical structures also exist in the native channel as proposed by recent models. The results are discussed in terms of proposals that various regions of membrane proteins participate in driving folding or oligomerization of the parent molecules.

Amino Acid Sequence↗

PubMed1995

The assembly and organization of the alpha 5 and alpha 7 helices from the pore-forming domain of Bacillus thuringiensis delta-endotoxin. Relevance to a functional model.

The pore-forming domain of Bacillus thuringiensis insecticidal CryIIIA delta-endotoxin contains two helices, alpha 5 and alpha 7, that are highly conserved within all different Cry delta-endotoxins. To gain information on the mode of action of delta-endotoxins, we have used a spectrofluorimetric approach and characterized the structure, the organization state, and the ability to self-assemble and to co-assemble within lipid membranes of alpha 5 and alpha 7. Circular dichroism (CD) spectroscopy revealed that alpha 7 adopts a predominantly alpha-helical structure in methanol, similar to what has been found for alpha 5, and consistent with its structure in the intact molecule. The hydrophobic moment of alpha 7 is higher than that calculated for alpha 5; however, alpha 7 has a lesser ability to permeate phospholipids as compared to alpha 5. Binding experiments with 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labeled peptide demonstrated that alpha 7 binds to phospholipid vesicles with a partition coefficient in the order of 10(4) M-1 similar to alpha 5, but with reduced kinetics and in a noncooperative manner, as opposed to the fast kinetics and cooperativity found with alpha 5. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/acceptor (rhodamine) peptides revealed that, in their membrane-bound state, alpha 5 self-associates but alpha 7 does not, and that alpha 5 coassembles with alpha 7 but not with an unrelated membrane bound alpha-helical peptide. Furthermore, resonance energy transfer experiments, using alpha 5 segments, specifically labeled in either the N- or C-terminal sides, suggest a parallel organization of alpha 5 monomers within the membranes. Taken together the results are consistent with an umbrella model suggested for the pore forming activity of delta-endotoxin (Li, J., Caroll, J., and Ellar, D. J. (1991) Nature 353, 815-821), where alpha 5 has transmembrane localization and may be part of the pore lining segment(s) while alpha 7 may serve as a binding sensor that initiates the binding of the pore domain to the membrane.

4-Chloro-7-nitrobenzofurazan↗

PubMed1995

Mechanisms for the modulation of membrane bilayer properties by amphipathic helical peptides.

The amphipathic helix, in which hydrophobic and hydrophilic residues are grouped on opposing faces, is a structural motif found in many peptides and proteins that bind to membranes. One of the physical properties of membranes that can be altered by the binding of amphipathic helices is membrane monolayer curvature strain. Class A amphipathic helices, which are present in exchangeable plasma lipoproteins, can stabilize membranes by reducing negative monolayer curvature strain; proline-punctuated class A amphipathic helical segments are particularly effective in this regard. This property is suggested to be associated with some of the beneficial biological effects of this protein. On the other hand, lytic amphipathic helical peptides can act by increasing negative curvature strain or by forming pores composed of helical clusters. Thus, different amphipathic helical peptides can be membrane stabilizing or be lytic to membranes, depending on the structural motif of the helix, which in turn determines the nature of its association with membranes. Features of these peptides that are responsible for their specific properties are discussed.

Amino Acid Sequence↗

PubMed1995

Molecular recognition between membrane-spanning polypeptides.

Integral membrane proteins have recently been shown to recognize and interact with other proteins within the membrane, either mimicking or altering their function, and with the lipid bilayer itself, resulting in a reorganization of native membrane protein. Membrane proteins are difficult to study using conventional methods such as X-ray crystallography, and so both synthesized and naturally occurring segments of membrane proteins have been used in the assessment of the mechanisms involved in their structure and organization.

Anti-Bacterial Agents↗

PubMed1994

The alpha-5 segment of Bacillus thuringiensis delta-endotoxin: in vitro activity, ion channel formation and molecular modelling.

A peptide with a sequence corresponding to the highly conserved alpha-5 segment of the Cry delta-endotoxin family (amino acids 193-215 of Bacillus thuringiensis CryIIIA [Gazit and Shai (1993) Biochemistry 32, 3429-3436]), was investigated with respect to its interaction with insect membranes, cytotoxicity in vitro towards Spodoptera frugiperda (Sf-9) cells, and its propensity to form ion channels in planar lipid membranes (PLMs). Selectively labelled analogues of alpha-5 at either the N-terminal amino acid or the epsilon-amine of its lysine, were used to monitor the interaction of the peptides with insect membranes. The fluorescent emission spectra of the 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labelled alpha-5 peptides displayed a blue shift upon binding to insect (Spodoptera littoralis) mid-gut membranes, reflecting the relocation of the fluorescent probes to an environment of increased apolarity, i.e. within the lipidic constituent of the membrane. Moreover, midgut membrane-bound NBD-labelled alpha-5 peptides were protected from enzymic proteolysis. Functional characterization of alpha-5 has revealed that it is cytotoxic to Sf-9 insect cells, and that it forms ion channels in PLMs with conductances ranging from 30 to 1000 pS. A proline-substituted analogue of alpha-5 is less cytolytic and slightly more exposed to enzymic digestion. Molecular modelling utilizing simulated annealing via molecular dynamics suggests that a transbilayer pore may be formed by alpha-5 monomers that assemble to form a left-handed coiled coil of approximately parallel helices. These findings further support a role for alpha-5 in the toxic mechanism of delta-endotoxins, and assign alpha-5 as one of the transmembrane helices which form the toxic pore. The suggested role is consistent with the recent finding that cleavage of CryIVB delta-endotoxin in a loop between alpha-5 and alpha-6 is highly important for its larvicidal activity [Angsuthanasombat, Crickmore and Ellar (1993) FEMS Microbiol. Lett. 111, 255-262].

4-Chloro-7-nitrobenzofurazan↗

PubMed1994

Capacities of pardaxin analogues to induce fusion and leakage of negatively charged phospholipid vesicles are not necessarily correlated.

Peptide-induced vesicle fusion is frequently accompanied by leakage of vesicle contents. To determine the correlation between these two processes, we studied the interaction of the amphiphilic peptide pardaxin and two of its analogues with large unilamellar vesicles composed of phosphatidylserine. A pardaxin analogue with a positive charge at both its C- and N-termini induced significantly more fusion but less leakage than the parent peptide. Fusion and leakage were studied with large unilamellar vesicles of two sizes. Aggregation of vesicles was found to be the rate-limiting step in the overall fusion process induced by the peptides. The rates and extents of fusion, determined by membrane mixing, increase in vesicle size, and mixing of aqueous contents, were significantly enhanced in the presence of 2.5-5 mM Mg2+ which promoted vesicle aggregation. Model calculations showed that increasing the peptide to lipid ratio resulted in a parallel increase in the fusion rate constants. As the average vesicle diameter was increased, the extent of leakage was enhanced, as more peptide molecules were bound to each vesicle. The mode of leakage induced by the peptides was also investigated. Our results suggest that the potency of a peptide to induce vesicle fusion is not necessarily associated with its capacity to induce leakage, and we further elucidate how these capacities depend on the structures of the peptides.

Amino Acid Sequence↗

PubMed1994

Spectrum of antimicrobial activity and assembly of dermaseptin-b and its precursor form in phospholipid membranes.

Dermaseptins are 27-34 amino acid antimicrobial peptides that irreversibly inhibit growth of pathogenic filamentous fungi, in addition to their ability to inhibit the growth of bacteria, yeasts, and protozoa. Synthetic peptides, with sequences corresponding to dermaseptin-b (DS-b) and its N-terminal extended precursor form dermaseptin-B (DS-B), were synthesized and investigated with respect to their spectrum of antimicrobial activity and their mode of interaction with model membranes composed of PS or PC/PS phospholipids. We found that DS-B is much more potent than DS-b against all microorganisms tested. Furthermore, despite significant structural identity between DS-b and DS-S (Pouny et al., 1992), only the former is highly effective at inhibiting the growth of filamentous fungi. The peptides were labeled selectively at their N-terminal amino acid with either 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or rhodamine fluorescent probes, which facilitated the determination of their partition coefficients with phospholipid membranes and their organization in their membrane-bound state. The partition coefficients of DS-B are 10-fold higher than those of DS-b and DS-S, with both acidic and zwitterionic phospholipid vesicles. This may explain the ability of DS-B to permeate both types of vesicles efficiently. Furthermore, while both DS-b and DS-B interact with phospholipid membranes in a noncooperative manner, they are self-associated in their membrane-bound state. This noncooperative binding probably prevents aggregation of the peptides on the surface of outer bacterial membranes, and assists them in efficiently diffusing into the inner target membranes. The exceptional property of DS-B to bind strongly to phospholipid membranes and to form small bundles correlates with its high potential to kill yeast and filamentous fungi. As a molecular model, dermaseptins may be of potential interest in drug design, particularly in antifungal warfare.

Amino Acid Sequence↗

PubMed1994

Mode of action of the antibacterial cecropin B2: a spectrofluorometric study.

Cecropin B2 (CecB) is a 35 amino acid residue, antibacterial peptide that was isolated from the hemolymph and cuticular matrix of the silkworm, Bombyx mori. Synthetic peptides with sequences corresponding to CecB and its truncated analogue, [3-->35]CecB, were synthesized and selectively labeled at their N-terminal amino acids with either 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or rhodamine fluorescent probes. Utilization of these probes facilitated study of the interaction of cecropin with model phospholipid membranes at a high lipid/peptide molar ratio (approximately 3000:1), permitting investigation of the initial steps involved in this process. The surface partition coefficient of CecB, derived from binding isotherms of the NBD-labeled peptide, was 10-fold higher with acidic phospholipids than with zwitterionic ones, which correlates with the high efficiency of CecB and its analogues in permeating acidic phospholipid vesicles. Furthermore, a direct correlation was found between the antibacterial activity of CecB or its truncated analogues and the ability of their Rho-labeled analogues to interact with bacteria and human red blood cells. We propose that CecB binds phospholipid membranes preferentially as monomers lying on the surface, rather than cooperatively as bundles that form transmembranal pores via a "barrel stave" mechanism. This is based on the following: (i) the linearity of CecB's binding isotherms; (ii) the low energy transfer between membrane-embedded donor and acceptor-labeled CecB, even in the presence of a transmembrane potential; (iii) the surface localization of CecB's N-terminus; (iv) the need for more than 100 peptide molecules per phospholipid vesicle to induce initial ion leakage; and (v) the fact that CecB is a highly positively charged amphipathic alpha-helix, and therefore it is not expected to transverse the membrane as a monomer. We speculate that the non-cooperative binding of the peptides on the outer surface of the bacteria (i.e., no aggregation of CecB monomers) may help them to diffuse efficiently into the inner membrane, which is thought to be the target of antibacterial peptides.

Amino Acid Sequence↗

PubMed1994

Synthetic S-2 and H-5 segments of the Shaker K+ channel: secondary structure, membrane interaction, and assembly within phospholipid membranes.

Current models of voltage-activated K+ channels predict that the channels are formed by the coassembly of four polypeptide monomers, each of which consists of six transmembrane segments (S1-S6) and long terminal domains. The aqueous pores are thought to be composed of the conserved H-5 regions contributed by four monomers. In this study, two putative membrane-embedded segments of the Shaker K+ channel were synthesized. One segment corresponds to the putative, transmembrane helix S-2 (amino acids 275-300), and the other corresponds to the highly conserved 12 amino acid residues within the H-5 region [amino acids 432-443, designated (12)H-5]. Structural and functional characterization at elevated lipid/peptide molar ratios (> 3000:1) was performed on the two segments, as well as on a previously synthesized 21 amino acid long peptide with a sequence resembling the entire H-5 region (designated (21)H-5) (Peled & Shai, 1993). Circular dichroism spectroscopy revealed that S-2 adopts predominantly alpha-helical structure in both trifluoroethanol and 35 mM SDS (78% or 99%, respectively), while (12)H-5 and (21)H-5 adopt low alpha-helical structure only in the presence of 35 mM SDS. Functional characterization demonstrated that S-2 and (12)H-5 segments bind to zwitterionic phospholipids, with partition coefficients on the order of 10(4) M-1. Resonance energy transfer measurements, between donor/acceptor-labeled pairs of peptides, revealed that the peptides self-associate in their membrane-bound state, which may correlate with the existence of functional interactions between the conserved (12)H-5 regions of different subunits of K+ channels (Kirsch et al., 1993).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Secondary structure and membrane localization of synthetic segments and a truncated form of the IsK (minK) protein.

IsK, also referred to as minK, is a membrane protein consisting of 130 amino acids and localized mainly in epithelial cells but also in human T lymphocytes. Depending on the cRNA concentration that was injected into Xenopus oocytes, IsK and its truncated forms can induce either a K+ current alone or both K+ and Cl- currents [Attali et al. (1993) Nature 365, 850-852]. To obtain information on the secondary structure and the topology of IsK in a membrane-bound state, the synthesis, fluorescent-labeling, and structural and functional characterization of five polypeptides of 20-63 amino acids within the rat IsK protein were examined. The alpha-helical content of the segments, assessed in methanol using circular dichroism, suggests that both the N-terminal and transmembrane segments of IsK adopt alpha-helical structures. Binding experiments and the blue shift of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled peptides suggest that while both the alpha-helical transmembrane segment and the N-terminal of IsK are located within the lipid bilayer, the linking segment between the two segments lies on the surface of the membrane. The fluorescence energy transfer, between donor and acceptor-labeled truncated IsK, suggests that it aggregates within phospholipid membranes. Although a protein whose sequence is similar to that of truncated IsK can induce K+ channel activity when expressed in Xenopus oocytes, the inability of a truncated IsK to form functional K+ channels in planar lipid membranes supports increasing evidence that the protein alone cannot form a K+ channel.

4-Chloro-7-nitrobenzofurazan↗

PubMed1994

Interaction of fluorescently labeled analogues of the amino-terminal fusion peptide of Sendai virus with phospholipid membranes.

A peptide representing the NH2-terminal (33 amino acid residues) of the fusion protein (F) of Sendai virus, as well as its Gly12-->Ala12 mutant, were synthesized, fluorescently labeled, and spectroscopically and functionally characterized. Peptide-induced vesicle fusion was demonstrated by a combination of increased visible absorbance, lipid mixing assay, and electron microscopy. Both peptides, with the mutant peptide being significantly more potent, were shown to induce membrane fusion and bilayer perturbation of negatively charged phospholipid vesicles. These results are consistent with a previous study that showed that a similar mutation in the homologous NH2-terminal segment of simian virus 5 greatly enhanced syncytium formation (Horvath, C. M., and Lamb, R. A. (1992) J. Virol. 66, 2443-2455). Circular dichroism spectroscopy revealed similar high alpha-helical contents of both peptides in methanol and in trifluoroethanol. Using fluorescently labeled peptide analogues we found that (i) the peptides' membrane partition coefficients are in the range of 10(5) M-1; (ii) the NH2 terminus of the wild-type peptide is located within the lipid bilayer, whereas that of the variant peptide lies on the surface; and (iii) both peptides tend to self-associate in their membrane-bound state. The results support a model in which an alpha-helical secondary structure and self-aggregation of peptides are necessary conditions for membrane fusion. The observed differences in the peptides' fusogenic abilities are hypothesized to result from differences in the peptides' degree of penetration into the membrane, induction of membrane destabilization, and ability to cause vesicles to aggregate. The data support Sendai virus-cell fusion models in which the fusion peptide plays a crucial role in fusion induction by destabilizing the bilayer and by triggering the association of viral fusion protein molecules.

Amino Acid Sequence↗

PubMed1994

Pardaxin: channel formation by a shark repellant peptide from fish.

The results of the various studies describing the mechanism involved in pore formation by pardaxin and some of its analogues, support a 'barrel-stave' model (Ehrenstein amd Lecar, 1977). In this model pardaxin exerts its activity via three successive steps: (i) a fast binding step (as reflected by the rapid increase of NBD fluorescence in the presence of vesicles); (ii) insertion of peptides into the lipid bilayer; and (iii) the monomers aggregate into a barrel-like formation in which a central aqueous pore surrounded by proteins is formed. This pore increases in diameter through the progressive recruitment of additional monomers. Both the fluorescence energy transfer (FET) studies and the observation of a significant difference in the increase of NBD fluorescence, depending on which terminal was labelled by the fluorophore, support a model by which aggregates are formed in an ordered parallel manner, where the C-terminus is more exposed to the aqueous phase.

Amino Acid Sequence↗

PubMed1993

Structural characterization, membrane interaction, and specific assembly within phospholipid membranes of hydrophobic segments from Bacillus thuringiensis var. israelensis cytolytic toxin.

The Bacillus thuringiensis var. israelensis (Bti) cytolytic toxin is hypothesized to exert its toxic activity via pore formation in the cell membrane as a result of the aggregation of several monomers. To gain insight into the toxin's mode of action, 2 putative hydrophobic 22 amino acid peptides were synthesized and characterized spectroscopically and functionally. One peptide corresponded to the putative amphiphilic alpha-helical region (amino acids 110-131, termed helix-2), and the other to amino acids 50-71 (termed helix-1) [Ward, E. S., Ellar, D. J., & Chilcott, C. N. (1988) J. Mol. Biol. 202, 527-535] of the toxin. Circular dichroism spectroscopy revealed that both segments adopt high alpha-helical content in a hydrophobic environment, in agreement with previous models. To monitor peptide-lipid and peptide-peptide interactions, the peptides were labeled selectively with either 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) (to serve as donor) or tetramethylrhodamine (to serve as an acceptor), at their N-terminal amino acids. Both segments bind strongly to small unilamellar vesicles, composed of zwitterionic phospholipids, with surface partition coefficients on the order of 10(4) M-1. The shape of the binding isotherms indicates that helix-2 forms large aggregates within phospholipid membranes. Resonance energy transfer experiments demonstrated that the segments self-associate and interact with each other, but do not associate with unrelated membrane-bound peptides. Functional characterization demonstrated that helix-2 permeates phospholipid SUV with a potency similar to that of naturally occurring pore-forming peptides. Thus, the results support a role for helices-1 and -2 in the assembly and in the pore formation by Bti toxin.

Amino Acid Sequence↗

PubMed1993

Membrane interaction and self-assembly within phospholipid membranes of synthetic segments corresponding to the H-5 region of the shaker K+ channel.

The voltage-activated K+ channels are assumed to be formed by the coassembly of four polypeptide monomers. Each of these monomers is postulated to consist of six transmembrane segments (S1 to S6), and long N- and C-terminal domains. The highly conserved linker, H-5, between the fifth and the sixth transmembrane segments, is hypothesized to line the lumen of the K+ channel formed by the bundle of the transmembrane segments of the monomers. Herein we utilize the spectrofluorometric approach and investigate the interaction with phospholipid membranes of fluorescently-labeled synthetic peptides, whose sequences are derived from the H-5 region. Binding experiments reveal that the peptides can strongly bind to phospholipid membranes with partition coefficients on the order of 10(4) M-1. However, a truncated peptide without four amino acids within the most conserved region (amino acids 432-435) did not bind to the membranes at all. Moreover, the single substitution of a conserved tryptophan at position 435 to serine reduced the partition coefficient of the peptide approximately 5-fold, which may account for a mutated K+ channel with this substitution not producing functional channels (Yool & Schwarz, 1991). Structural characterization using circular dichroism spectroscopy (CD) reveals that H-5 can partially adopt an alpha-helix structure in hydrophobic environments. Resonance energy transfer (RET) experiments reveal that the H-5-derived segments can self-assemble within the membrane but cannot coassemble with other unrelated membrane-bound peptides. The results herein support the hypothesis that H-5 segments are packed in close proximity and might participate in mediating the appropriate assembly of the core region of K+ channel monomers.

Amino Acid Sequence↗

PubMed1993

Synthetic peptides corresponding to the calmodulin-binding domains of skeletal muscle myosin light chain kinase and human erythrocyte Ca2+ pump interact with and permeabilize liposomes and cell membranes.

Synthetic calmodulin-binding (CaM-binding) peptides (CBPs) representing CaM-binding domains of Ca2+/CaM-dependent enzymes have been reported to interfere with the activity of the melanocyte-stimulating hormone (MSH) receptor function in melanoma cells [Gerst, J. E., & Salomon, Y. (1988) J. Biol. Chem. 263, 7073-7078]. We postulated that membrane lipids may play an important role in the mode of action of CBPs on cells. We therefore tested the ability of CBPs to interact with membrane bilayers. Using artificial phospholipid vesicles, or M2R melanoma cells and cell membranes derived therefrom, as models, we report here that synthetic peptides representing the CaM-binding domains of skeletal muscle myosin light chain kinase (M5) and the human erythrocyte calcium pump (C28W), as well as other CBPs, interact with lipid bilayers and cell membranes. Significant interactions of CBPs with the lipid bilayer were detected in both model systems. M5 and C28W were found to partition into the lipid bilayer of melanoma cell membranes and soybean lecithin vesicles, and surface partition constants obtained (for the liposome model) were in the range 10(3)-10(4) M-1. In addition, C28W and its N-modified NBD derivative were found to inhibit [125I]iodo-[Nle4,D-Phe7]alpha MSH binding to cultured M2R melanoma cells. These and other CBPs were also found to induce the release of cations and calcein from liposomes, suggesting that the interaction of CBPs with the lipid bilayer increases membrane permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

pH- and ionic strength-dependent fusion of phospholipid vesicles induced by pardaxin analogues or by mixtures of charge-reversed peptides.

The fusogenic properties of the neurotoxin paradaxin and eight of its analogues with small unilamellar vesicles (SUV), composed of egg phosphatidylcholine and phosphatidylserine (PC/PS), were investigated. Fusion was demonstrated by a lipid-mixing assay and by an increase in vesicle size as revealed by electron microscopy. The lipid-mixing assay was performed at either neutral (pH 6.8) or acidic (pH 4.5) conditions, in solutions containing either high or low salt concentrations. A low level of fusion could be induced at neutral pH only by pardaxin derivatives with amino groups at both the peptide's backbone and N-terminus. However, a marked enhancement in the fusogenic activity occurred when amino groups were present also in the C-terminus. Pardaxin analogues in which amino groups were substituted by carboxylic groups induced elevated levels of fusion only at high salt concentrations where enhancement of aggregation occurs, and acidic pH, which increased alpha-helicity. The influence of mutual interactions between pardaxin's analogues possessing complementary charges on the lipid-mixing process was also studied. At neutral pH and high salt, an inactive acidic analogue increased the fusogenic activity of a complementary-charged basic peptide. However, such mutual interactions at low salt concentrations reduced the fusogenic activity of the pardaxin analogues. Analogues containing D-amino acids were not fusogenic, thus demonstrating the structural specificity of these observations. The results indicate that the charge, alpha-helical structure, and aggregation of peptide monomers play an important role in the fusogenic ability of polypeptides.

Amino Acid Sequence↗

PubMed1993

Structural and functional characterization of the alpha 5 segment of Bacillus thuringiensis delta-endotoxin.

One of the most conserved sequences in various delta-endotoxins is the 30 amino acid long block I. Block I of cryIIIA delta-endotoxin contains a 23 amino acid amphiphilic alpha-helix termed alpha 5. The potential involvement of this alpha 5 helix in the toxic mechanism of delta-endotoxin was examined. For this purpose, a peptide corresponding to the alpha 5 segment and its proline incorporated analogue (P-alpha 5) were synthesized and characterized. The alpha-helical content of the peptides, assessed in methanol by circular dichroism (CD), was 58% and 24% for alpha 5 and P-alpha 5, respectively. To monitor the interaction of alpha 5 peptides with phospholipid membranes, they were selectively labeled at their N-terminal amino acids with the fluorescent probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or carboxyfluorescein. Fluorometric studies allowed the calculation of membrane surface partition constants, which were about 10(4) M-1 for both alpha 5 and P-alpha 5, and revealed that their N-terminals are located within the lipid bilayers. The shape of the binding isotherms indicated that alpha 5 aggregated in both zwitterionic and acidic vesicles. Functional characterization of the alpha 5 peptides was determined by assessing their ability to dissipate a diffusion potential from sonicated small unilamellar vesicles (SUV) composed of zwitterionic or acidic phospholipids and to lyse human erythrocytes. alpha 5 was much more active than P-alpha 5 in both assays. Moreover, membrane-bound alpha 5 was more protected from enzymatic proteolysis than P-alpha 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Spectroscopic and functional characterization of the putative transmembrane segment of the minK potassium channel.

MinK (Isk) is a voltage-dependent K+ channel whose gene has been recently cloned and which consists of 130 amino acids [Takumi, T., Ohkubo, H., & Nakanishi, S. (1988) Science 242, 1042-1045]. The protein contains one putative transmembrane segment by hydropathy analysis. Whether this putative transmembrane segment is involved in the function of the protein was studied. A 32 amino acid peptide (residues 41-72) with the sequence SKLEALYILMVLGFFGFFTLGIMLSYIRSKKL, containing the hypothesized transmembrane domain, designed TM-minK, was synthesized and fluorescently labeled. The alpha-helical content of TM-minK, assessed in methanol using circular dichroism (CD), was 57%. The fluorescent emission spectrum of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled TM-minK displayed a blue shift upon binding to small unilamellar vesicles (SUV), reflecting a relocation of the fluorescent probe to an environment of increased apolarity, i.e., within the lipid bilayer. The increase in NBD's fluorescence upon mixing NBD-labeled TM-minK with small unilamellar vesicles (SUV) was used to generate a binding isotherm, from which was derived a surface partition coefficient of 5.5 x 10(4) M-1. Fluorescence energy transfer measurements between carboxyfluoresceine-labeled and rhodamine-labeled analogues suggest that TM-minK aggregates within membranes. In addition, single-channel experiments revealed that TM-minK can form single channels in planar lipid membranes only when a trans negative potential is applied. The findings herein experimentally support a role of the transmembrane segment of minK both in the assembly and as a constituent of the pore formed by the protein.

4-Chloro-7-nitrobenzofurazan↗