RESUMO
The single channel data for 4 different linear gramicidins containing either 4 Trp, 4 Phe, 4 Tyr or TyrBzl have been analyzed on the basis of 3 barriers-2 sites model. They form 2 families which differ by their single channel behavior and thus different energy profiles of the channel. A relationship between the surface potential and the entry barrier is proposed.
Assuntos
Gramicidina , Canais Iônicos , Condutometria , Metabolismo Energético , Computação Matemática , Potenciais da Membrana , Membranas Artificiais , Relação Estrutura-AtividadeRESUMO
It is shown that 2 cyclic tetrapeptides, namely tentoxin and HC toxin, are able to induce the formation of transmembrane ionic channels, although a carrier mechanism could be expected on the basis of their chemical structure (presence of proline or N-methylated residues). Since other cyclic peptides but of larger size, i.e., tyrocidines, gramicidin S (decapeptides) and an octapeptide with a sequence similar to that of HC toxin, are also able to form pores, it appears that this property can be extended to a large number of cyclic peptides. A pore structure based on aggregates is proposed.
Assuntos
Canais Iônicos/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Dicroísmo Circular , Eletrofisiologia , Bicamadas Lipídicas , Modelos QuímicosRESUMO
The release of cytochrome c from mitochondria to the cytosol is a crucial step of apoptosis that involves interactions of Bax and tBid proteins with the mitochondrial membrane. We investigated Bax and tBid interactions with (i) phosphatidylcholine (PC) monolayer as the main component of the outer leaflet of the outer membrane, (ii) with phosphatidylethanolamine (PE) and phosphatidylserine (PS) that are present in the inner leaflet and (iii) with a mixed PC/PE/Cardiolipin (CL) monolayer of the contact sites between the outer and inner membranes. These interactions were studied by measuring the increase of the lipidic monolayer surface pressure induced by the proteins. Our measurements suggest that tBid interacts strongly with the POPC/DOPE/CL, whereas Bax interaction with this monolayer is about 12 times weaker. Both tBid and Bax interact moderately half as strongly with negatively charged DOPS and non-lamellar DOPE monolayers. TBid also slightly interacts with DOPC. Our results suggest that tBid but not Bax interacts with the PC-containing outer membrane. Subsequent insertion of these proteins may occur at the PC/PE/CL sites of contact between the outer and inner membranes. It was also shown that Bax and tBid being mixed in solution inhibit their insertion into POPC/DOPE/CL monolayer. The known 3-D structures of Bax and Bid allowed us to propose a structural interpretation of these experimental results.
Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Metabolismo dos Lipídeos , Proteína X Associada a bcl-2/metabolismo , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Cálcio/fisiologia , Cardiolipinas/química , Cardiolipinas/metabolismo , Bovinos , Humanos , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Estrutura Terciária de Proteína , Proteína X Associada a bcl-2/químicaRESUMO
In the field of drug delivery there has been a continuous study of powerful delivery systems to aid non permeable drugs in reaching their intracellular target. Among the systems explored are cell penetrating peptides (CPPs), which first garnered interest a decade ago when the interesting translocation properties of the pioneer CPPs Tat and Antp were described. A new family of CPPs has recently been described as non cytotoxic Pro-rich vectors with favorable profiles for internalization in HeLa cells. Fatty acyl moieties that can tune a peptide's interaction with the lipophilic environment of a cell membrane have been incorporated into the Pro-rich sequence. Improvements in cellular uptake of peptides modified with fatty acyl groups, as studied by confocal microscopy and flow cytometry, as well as the results obtained by the interaction of these peptides with a model dioleoylphosphatidylcholine (DOPC) membrane and transmission electron microscopy (TEM), illustrate the importance of the fatty acyl moieties for efficient internalization.
Assuntos
Ácidos Graxos/metabolismo , Peptídeos/metabolismo , Prolina/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Ácidos Graxos/química , Citometria de Fluxo , Células HeLa , Humanos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Peptídeos/síntese química , Peptídeos/química , Fosfatidilcolinas , Prolina/química , Domínios Proteicos Ricos em ProlinaRESUMO
A comparative monolayer and infrared study of analogues of gramicidin A containing either tyrosines or naphthylalanines instead of tryptophans indicates that the nature of the aromatic residues influences the favoured conformation of the peptides. Polar residues favour the single stranded IIDL helix while non polar residues favour the double stranded helix. For partly tryptophan to naphthylalanine substituted analogues the positions of the substitutions orientate the favored conformation. The nature of these substitutions may also modify the peptide-lipid interactions.
Assuntos
Gramicidina/química , Alanina/análogos & derivados , Alanina/química , Sequência de Aminoácidos , Bicamadas Lipídicas , Modelos Químicos , Conformação Molecular , Dados de Sequência Molecular , Espectrofotometria Infravermelho , Tirosina/químicaRESUMO
The behavior of two gramicidins incorporated into lipid monolayers is analyzed on the basis of the force and surface potential area curves. It is shown that the position of the gramicidins (helical axis parallel or perpendicular to the interface) depends on the monolayer pressure and that these molecules are not miscible with dioleoylphosphatidylcholine. Surface potential measurements suggest the existence of a relationship between the single channel characteristics and the surface potential and indicate that the tryptophans are essential for lowering the lipid surface potential in agreement with the single channel behaviour of both gramicidin A and gramicidin M.
Assuntos
Gramicidina , Canais Iônicos/fisiologia , Lipossomos , Modelos Teóricos , Fosfatidilcolinas , Potenciais da Membrana , Pressão , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
We describe the design and the Fmoc/tBu solid phase synthesis of a 20 residue long peptide containing five regularly distributed lysines. Cyclization of this peptide was achieved using BOP as coupling agent. After side-chain deprotection, all the basic residues were iodoacetylated and then allowed to react either with a C-terminal free COOH peptide or with peptides bearing a cysteamide group. The final pentameric templates were identified by mass and amino acid analysis which gave data compatible with the expected values.
Assuntos
Canais Iônicos/síntese química , Oligopeptídeos/síntese química , Moldes Genéticos , Acetilação , Sequência de Aminoácidos , Canais Iônicos/química , Lisina/química , Dados de Sequência Molecular , Oligopeptídeos/química , Peptídeos Cíclicos/síntese química , Conformação Proteica , Sulfetos/químicaRESUMO
Three different gramicidin A analogues bearing acyl chains of various length on the ethanolamine moiety have been studied by investigating their single channel behaviour and their monolayer properties. It is shown that the single channel conductance does not depend on the substitution of the ethanolamine OH group and that the channel lifetime is roughly proportional to the length of the alkyl chain. The monolayer study indicates that acylation of gramicidin A produces compounds which have medium-dependent conformations. These acylated compounds are miscible with lipids, while GA is not, and the surface potential is not modified by the esterification of the alcohol group.
Assuntos
Gramicidina/química , Acilação , Sequência de Aminoácidos , Fenômenos Biofísicos , Biofísica , Condutividade Elétrica , Dados de Sequência Molecular , Espectrofotometria Infravermelho , Propriedades de SuperfícieRESUMO
Increasing evidence suggests that the HIV envelope binds through its surface (SU) gp120 not only to receptors and coreceptors, but also to other components of the cellular membrane where the glycolipids appear to be good candidates. To assess the ability of HIV-1 SU gp120 to penetrate into phospholipid membranes, we carried out a study of the interactions between a recombinant SU gp120 from HIV-1/HXB2 and artificial lipid monolayers mimicking the composition of the outer leaflet of the lymphocytes and which were spread at the air-water interface. We show that the protein, in its aggregated form, has amphipathic properties and that the insertion of this amphipathic species into lipids is favored by the presence of sphingomyelin. Furthermore, cholesterol enhances the penetration into mixed phosphatidylcholine-sphingomyelin monolayers. Coexistence of different physical states of the lipids and thus of domains appears to play a major role for protein penetration independently of the presence of receptors and coreceptors.
Assuntos
Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/patogenicidade , Lipídeos de Membrana/metabolismo , Adsorção , Animais , Bovinos , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/virologia , Colesterol/metabolismo , Gangliosídeo G(M3)/metabolismo , HIV-1/fisiologia , Humanos , Técnicas In Vitro , Lipídeos de Membrana/química , Membranas Artificiais , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
To better understand the nature of the mechanism involved in the membrane uptake of a vector peptide, the interactions between dioleoylphosphatidylcholine and a primary amphipathic peptide containing a signal peptide associated with a nuclear localization sequence have been studied by isotherms analysis of mixed monolayers spread at the air-water interface. The peptide and the lipid interact through strong hydrophobic interactions with expansion of the mean molecular area that resulted from a lipid-induced modification of the organization of the peptide at the interface. In addition, a phase separation occurs for peptide molar fraction ranging from about 0.08 to 0.4 Atomic force microscopy observations made on transferred monolayers confirm the existence of phase separation and further reveal that mixed lipid-peptide particles are formed, the size and shape of which depend on the peptide molar fraction. At low peptide contents, round-shaped particles are observed and an increase of the peptide amount, simultaneously to the lipidic phase separation, induces morphological changes from bowls to filamentous particles. Fourier transform infrared spectra (FTIR) obtained on transferred monolayers indicate that the peptide adopts a beta-like structure for high peptide molar fractions. Such an approach involving complementary methods allows us to conclude that the lipid and the peptide have a nonideal miscibility and form mixed particles which phase separate.
Assuntos
Lipídeos/química , Peptídeos/química , Tensoativos/química , Microscopia de Força Atômica , Fosfatidilcolinas/química , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
To improve the understanding of the membrane uptake of an amphipathic and positively charged vector peptide, we studied the interactions of this peptide with different phospholipids, the nature of whose polar headgroups and physical states were varied. Three lipids were considered: dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and dioleoylphosphatidylglycerol (DOPG). The approach was carried out by three complementary methods: compression isotherms of monolayers and atomic force microscopy observations associated with Fourier transform infrared investigations. From analysis of the compression isotherms, it was concluded that the peptide interacts with all lipids and with an expansion of the mean molecular area, implying that both components form nonideal mixtures. The expansion was larger in the case of DOPG than for DPPC and DPPG because of an alpha to beta conformational transition with an increase in the peptide molar fraction. Atomic force microscopy observations showed that the presence of small amounts of peptide led to the appearance of bowl-like particles and that an increase in the peptide amounts generated the formation of filaments. In the case of DOPG, filaments were found at higher peptide molar fractions than already observed for DOPC because of the presence of negatively charged lipid headgroups.
Assuntos
Peptídeos/química , Fosfolipídeos/química , 1,2-Dipalmitoilfosfatidilcolina/química , Sequência de Aminoácidos , Dicroísmo Circular , Microscopia de Força Atômica , Conformação Molecular , Dados de Sequência Molecular , Fosfatidilgliceróis/química , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de SuperfícieRESUMO
Two peptides designed for drug delivery were generated by the combination of a signal peptide with a nuclear localization sequence and are shown to facilitate the cellular internalization of small molecules which are covalently linked to these peptides. In order to understand the mechanism of internalization, the conformations of the peptides were investigated through different approaches both in solution and in membrane-mimicking environments. These peptides are highly versatile and adopt different conformational states depending on their environment. While in a disordered form in water, they adopt an alpha-helical structure in TFE and in the presence of micelles of SDS or DPC. The structured domain encompasses the hydrophobic part of the peptides, whereas the charged C-termini remain unstructured. In contrast, in the presence of lipids and whatever the nature of the phosphate headgroup, the two peptides mainly adopt an antiparallel beta-sheet form and embed in the lipidic cores. This result suggests that the beta-sheet is responsible for the translocation through the cellular membranes but also questions the conformational state of signal peptides when associated to hydrophilic sequences.
Assuntos
Proteínas de Transporte/química , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Mimetismo Molecular , Sequência de Aminoácidos , Células Cultivadas , Dicroísmo Circular , Fibroblastos/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação Proteica , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The conformations of two peptides produced by the combinations of a nuclear localization sequence and a sequence issued from the fusion protein gp41 of HIV 1 have been analyzed both in solution and in membranes or in membrane mimicking environments. Both are shown to be nonordered in water, alpha-helical when incorporated into SDS micelles where the helical domain concerns the hydrophobic part of the peptides. Interactions with lipids induce the formation of beta-sheet and the lipid-peptide interactions are governed by the nature of the lipid polar headgroups. A monolayer study shows that replacement of the sequence separating the two sequences with an arginine favors the lipid-peptide interactions which may contribute to the understanding of the different, nuclear and membrane associated, cellular localizations of the peptides.
Assuntos
Proteína gp41 do Envelope de HIV/química , Membranas Artificiais , Peptídeos/química , Conformação Proteica , Sequência de Aminoácidos , Proteína gp41 do Envelope de HIV/metabolismo , Humanos , Dados de Sequência Molecular , Peptídeos/metabolismo , Fosfolipídeos/metabolismo , Análise Espectral , ÁguaRESUMO
The plasma membrane outer leaflet plays a key role in determining the existence of rafts and detergent-resistant membrane domains. Monolayers with lipid composition mimicking that of the outer leaflet of renal brush border membranes (BBM) have been deposited on mica and studied by atomic force microscopy. Sphingomyelin (SM) and palmitoyloleoyl phosphatidylcholine (POPC) mixtures, at molar ratios varying from 2:1 to 4:1, were phase-separated into liquid condensed (LC) SM-enriched phase and liquid expanded (LE) POPC-enriched phase. The LC phase accounted for 33 and 58% of the monolayers surface for 2:1 and 4:1 mixtures, respectively. Addition of 20-50 mol % cholesterol (Chl) to the SM/POPC (3:1) mixtures induced marked changes in the topology of monolayers. Whereas Chl promoted the connection between SM domains at 20 mol %, increasing Chl concentration progressively reduced the size of domains and the height differences between the phases. Lateral heterogeneity was, however, still present at 33 mol % Chl. The results indicate that the lipid composition of the outer leaflet is most likely responsible for the BBM thermotropic transition properties. They also strongly suggest that the common maneuver that consists of depleting membrane cholesterol to suppress rafts does not abolish the lateral heterogeneity of BBM membranes.
Assuntos
Rim/citologia , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Microvilosidades/química , Animais , Bovinos , Colesterol/metabolismo , Microdomínios da Membrana/ultraestrutura , Microscopia de Força Atômica , Microvilosidades/metabolismo , Microvilosidades/ultraestrutura , Fosfatidilcolinas/metabolismo , Esfingomielinas/metabolismoRESUMO
After activation, Bacillus thuringiensis (Bt) insecticidal toxin forms pores in larval midgut epithelial cell membranes, leading to host death. Although the crystal structure of the soluble form of Cry1Aa has been determined, the conformation of the pores and the mechanism of toxin interaction with and insertion into membranes are still not clear. Here we show that Cry1Aa spontaneously inserts into lipid mono- and bilayer membranes of appropriate compositions. Fourier Transform InfraRed spectroscopy (FTIR) indicates that insertion is accompanied by conformational changes characterized mainly by an unfolding of the beta-sheet domains. Moreover, Atomic Force Microscopy (AFM) imaging strongly suggests that the pores are composed of four subunits surrounding a 1.5 nm diameter central depression.