RESUMO
To study the influence of the conformation of polypeptidic macromolecules on the generation of T-cell epitopes, sequential polypeptides with an octamer repeat unit were designed and synthesized. They adopt mainly unordered and alpha-helical conformations. Among these polypeptides, those containing proline are fully or partly unordered, and are more effective at inducing T-cell proliferation than a proline-free very stable alpha-helical polypeptide. This extremely stable alpha-helical conformation, probably stabilized by aggregation, would enhance its stability against proteolytic processing.
Assuntos
Ativação Linfocitária/imunologia , Oligopeptídeos/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Epitopos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Prolina/imunologia , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Gramicidin P (a gramicidin A in which the ethanolamine C-terminus is replaced by methylamine) was synthesized and shown to have the same single-channel conductance behavior as gramicidin A. The results are discussed in connection with the energy profile computed in the presence of water in comparison with the corresponding profile for gramicidin A.
Assuntos
Gramicidina/síntese química , Cátions , Fenômenos Químicos , Físico-Química , Gramicidina/farmacologia , Canais Iônicos , Potenciais da Membrana/efeitos dos fármacos , Membranas Artificiais , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Peptides eluted from the MHC class I K(d) molecule are generally nonamers that display a strong preference for Tyr in position 2 and Ile or Leu in position 9. We investigated the binding ability of several synthetic peptides which did not fit this consensus motif. In our peptides, Tyr(2) was substituted by other amino acids, i.e. LeU, Ile or Met. These peptides were variants of the 252-260 K(d)-restricted peptide SYIPSAEKI derived from the Plasmodium berghei circumsporozoite protein. They bound to purified K(d) molecules in vitro with intermediate affinity. One of them was tested for in vivo stimulation of T cells and induced a cytotoxic response. These results demonstrate the importance of binding motif refinement to discover new binding characteristics and new ligands such as low-affinity peptides.
Assuntos
Antígenos H-2/metabolismo , Peptídeos/metabolismo , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Sequência Consenso , Cricetinae , Feminino , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/imunologia , Ligação ProteicaRESUMO
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
In order to understand the difference in single channel behavior of gramicidin A as compared to that of gramicidin M- which is the mirror image of gramicidin M (all four tryptophanyl residues substituted by phenylalanine), conformational investigations were made under several experimental conditions. It is shown that, when examined under identical conditions, both molecules adopt the same conformations which could be identified in dimethyl sulfoxide (DMSO) and chloroform. In DMSO the conformation is based on a succession of beta-turns while in chloroform gramicidin A and M- can adopt a dimeric hybrid structure: a double helix terminated by two single-stranded helices involving the N- and C-terminal parts, respectively. It is therefore concluded that the difference in the energy profile between both gramicidins which was deduced from the ion transfer data has its origin in the nature of the aromatic side chains.
Assuntos
Gramicidina , Canais Iônicos/fisiologia , Modelos Biológicos , Clorofórmio , Dimetil Sulfóxido , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , Espectrofotometria Infravermelho , Relação Estrutura-AtividadeRESUMO
The behavior of an analogue of gramicidin A in which all four tryptophanyl residues are substituted by phenylalanyl and which shows a strong voltage effect on the single channel conductance is analyzed on the basis of a 'three-barrier--two-site' model. It is shown that in the gramicidin family the side chains of some amino acids, in spite of their location, which point outside the channel can play a major role in the binding of ions in the channel and thus can significantly modify the energy profile of the channel.
Assuntos
Gramicidina , Canais Iônicos/fisiologia , Modelos Biológicos , Transporte Biológico , Condutividade Elétrica , Íons , CinéticaRESUMO
Examination of beta-carbons coordinates of seryl, aspartyl and histidyl residues in active sites of alpha-chymotrypsin and subtilisin BPN' shows that a close geometrical arrangement can be obtained in an antiparellel beta-structure. Therefore some polypeptides incorporating serine, aspartic acid and histidine, poly (Gly-Ser-Asp-His-Ala-Pro) and poly [(Asp-Leu-AsP-Leu)10, (His-Leu-Ser-Leu)1], and expected to have some tendency to give rise to an antiparallel beta-conformation, have been prepared and studied. The second polymer only adopts a fairly well-defined beta-structure in aqueous solution. Catalytic activities of these products towards p-nitrophenyl acetate are not improved as compared to histidine. However, kinetic pK of histidine side-chain depends markedly upon the nature of the product, owing probably to a hydrophobic environment effect.
Assuntos
Ácido Aspártico , Histidina , Nitrofenóis , Peptídeos , Serina , Sequência de Aminoácidos , Sítios de Ligação , Quimotripsina/metabolismo , Dicroísmo Circular , Peptídeos/síntese química , Conformação Proteica , Espectrofotometria Infravermelho , Relação Estrutura-Atividade , Subtilisinas/metabolismoRESUMO
To construct a possible model of hydrolytic enzymes, the sequential polypeptide H[Cys(Acm)-Ser-Phe-Glu-Glu]nOH, n = 3-110, was prepared by polycondensation of H-Cys(Acm)-Ser(But)-Phe-Glu(OBut)-Glu(OBut)-O Su/1-hydroxybenzotriazole. In a solid state the polypeptide material showed beta-conformation both before and after cleavage of t-butyl protecting groups. The pentapeptide unit was synthetized by the Merrifield method utilizing modifications as follows: Gel phase synthesis on less than 0.5% cross-linked copolystyrene (quasi dissolved state). Centrifugal reactor. Ddz-amino acids, deprotected by 5% trifluoroacetic acid/dichloromethane/15 min. 3-Nitrophthalic anhydride to suppress formation of false sequences. Continuous photometric control of the completion of all operations during synthesis and transesterfication, yielding Ddz-Cys(Acm)-Ser(But)-Phe-Glu-(OBut)-Glu(OBut)-OMe (0.827 g; 53%, m.p. 180-182 degrees).
Assuntos
Hidrolases/síntese química , Peptídeos/síntese química , Sequência de Aminoácidos , Hidrolases/análise , Espectroscopia de Ressonância Magnética , Peptídeos/análise , Espectrofotometria InfravermelhoRESUMO
Tyr(Bzl) and Tyr gramicidin A were prepared by the solid phase method using a 4-(oxymethyl)-Pam resin and Bpoc as alpha-amino-protecting group. The benzylated analog [Gr.T(Bzl)] was purified by chromatography on silica gel and then on LH60 Sephadex. Removal of benzyl groups was carried out by hydrogenolysis and the debenzylated derivative (Gr.T) was purified in the same way. Both gramicidins were checked and characterized by t.l.c., HPLC, circular dichroism, 1H n.m.r. and single channel measurements. CD spectra were found to be different for Gr.T(Bzl) and Gr.T and strongly dependent upon the solvent and the concentration. Single channel conductance of Gr. T is slightly lower than that of Gr.A (A Gr.T approximately equal to 0.7 A Gr.T).
Assuntos
Gramicidina/síntese química , Dicroísmo Circular , Indicadores e Reagentes , Canais Iônicos , Bicamadas Lipídicas , Espectroscopia de Ressonância Magnética , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The activity of poly (Cys-Ser-Phe-Glu-Glu) and related polypeptides as models of cysteine-proteases towards p-nitrophenylacetate was studied. The reaction leads to the formation of an S-acetylated form which proved to be quite stable, except at very high pH values. The presence of imidazole groups in the neighbourhood of sulfhydryl functions seems to result in an enhancement of the activity, which we attributed to a cooperative interaction. However, this interaction has no effect upon the S-deacylation rate. In the case of cysteine we found that the reaction with NPA occurred through a S to N acyl shift. Our results lead us to suggest that, unless convincing proofs of deacylation are given, the various models of cysteine-proteases studied so far do not behave as "true catalysts".
Assuntos
Cisteína , Nitrofenóis , Peptídeos , Compostos de Sulfidrila , Acilação , Cisteína/análogos & derivados , Concentração de Íons de Hidrogênio , Hidrólise , ImidazóisRESUMO
We have previously shown that the carrier polytuftsin obtained by polycondensation of tuftsin, a naturally occurring macrophage activator, increases significantly the antibody response against a linked B-epitope. In the present work, we have studied the influence of different cross-linking reagents on the quality of the conjugation and on the immune response, at both B-cell and T-cell levels. We observed that the cross-linking method used for coupling the B-epitope to the carrier influences the immune response. A hypothesis is put forward to explain the differences observed.
Assuntos
Linfócitos B/imunologia , Reagentes de Ligações Cruzadas/química , Antígenos de Superfície da Hepatite B/imunologia , Maleimidas/química , Polímeros/química , Precursores de Proteínas/imunologia , Succinimidas/química , Sulfetos/química , Tuftsina/química , Sequência de Aminoácidos , Animais , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Anticorpos Anti-Hepatite B/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Linfócitos T/imunologiaRESUMO
The specific interaction between biotin and avidin was exploited in the affinity purification of solid-phase synthesized peptide libraries. During peptide library synthesis, by means of the single-resin method in which coupling on variable positions is carried out using an equimolar mixture of amino acids, biotin was used to cap the unreacted amino groups remaining after coupling of the equimolar amino acid mixture. The following synthesis and deprotection procedures were performed as usual in tert,-butyloxycarbonyl chemistry. The purification of the peptide mixture containing N-biotinylated sequences was performed by affinity chromatography on an avidin-agarose column. The unwanted terminated sequences were retained in the avidin column while the purified peptide mixture was eluted as indicated by reverse-phase HPLC and MS analysis monitoring. The avidin column was regenerated and the biotinylated sequences were released under reversible denaturing conditions. The usefulness of biotinylation for peptide library purification is demonstrated here for the first time for a peptide mixture containing by-products that cannot be separated from the mixture by classical HPLC purification. This purification technique could be applied to all syntheses, presenting difficult reacting steps.
Assuntos
Cromatografia de Afinidade/métodos , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos/análise , Avidina , Biotina , Dados de Sequência Molecular , Peptídeos/síntese químicaRESUMO
Analysis of the single-channel behavior of an analogue of gramicidin A in which all four tryptophyl residues are substituted by phenylalanyl suggests that the nature of the side chains may play an important role in the ion translocation process. Indeed, while infrared spectroscopy indicates that both peptides have very similar backbone conformations, they have different single-channel characteristics. The unit conductance of the analogue is much smaller than that of the natural product. Moreover, contrary to gramicidin A, it is voltage dependent.
Assuntos
Gramicidina , Canais Iônicos/fisiologia , Lipossomos , Modelos Biológicos , Potenciais da Membrana , PotássioRESUMO
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
Poly(Lys-Tyr-Tyr-Lys) was synthesized by polycondensation of the tetrapeptide unit using paranitrophenyl esters. The conformation of poly(Lys-Tyr-Tyr-Lys) is very dependent on its environment. CD spectra in bulk are difficult to interpret owing to the contribution of Tyr residues, but from ir spectra it seems that poly(Lys-Tyr-Tyr-Lys) adopts preferentially an unordered conformation in water. Addition of salts induces a partial transition to a beta structure. The behavior is different at interfaces. When poly(Lys-Tyr-Tyr-Lys) is spread as a film on a water subphase, the shape of the compression isotherm curves is compatible with a stacking of two beta-sheets. On a KCl subphase, the polymer film is more expanded and more compressible, and the isotherm curve resembles that of a polymer in a random conformation. The analysis by CD and ir spectroscopy of transferred monolayers using the Langmuir-Blodgett technique allowed us to confirm and make these data more precise: on a water subphase the spectra are those of an antiparallel beta structure. At the interface of a saline solution the spectra are compatible with a mixture of random coil (largely) and a small content of beta structures.
Assuntos
Peptídeos/química , Conformação Proteica , Sequência de Aminoácidos , Dicroísmo Circular , Indicadores e Reagentes , Dados de Sequência Molecular , Peptídeos/síntese química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Little information is available correlating the structural properties of peptides with their immunogenicity in terms of responses via cytotoxic T lymphocytes (CTLs). The TT-NP6 chimeric peptide, consisting of two copies of a promiscuous T-helper epitope (T: residues 288-302 from the fusion protein of the measles virus) linked to the NP6 T-cytotoxic epitope (NP6: residues 52-60 from the nucleoprotein of measles virus) was able to induce virus-specific CTL responses in the absence of any adjuvant and hydrophobic component. The present work was undertaken to gain insight into structural features of the TT-NP6 peptide that may be important in optimizing the CTL immunogenicity of the peptide. Circular dichroism data, obtained in a buffer of physiological ionic strength and pH, strongly suggest a self-associated state for the peptide, which was confirmed by a sedimentation velocity experiment. However, helix association is accompanied by loss of overall helical content. Thermal-dependence studies show that the unfolding of self-associated alpha-helices is significantly more pronounced than the unfolding of isolated alpha-helices. Circular dichroism data, together with tryptic limited proteolysis, suggest the presence of a charged amino acid within the hydrophobic core. This study should provide a basis for engineering more effective immunogenic peptides against the measles virus by increasing the stability of the TT-NP6 peptide.
Assuntos
Epitopos/química , Vírus do Sarampo/química , Proteínas Recombinantes de Fusão/química , Proteínas do Core Viral/química , Proteínas Virais de Fusão/química , Sequência de Aminoácidos , Dicroísmo Circular , Temperatura Alta , Espectrometria de Massas , Vírus do Sarampo/imunologia , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fosfatos/farmacologia , Desnaturação Proteica , Estrutura Secundária de Proteína/efeitos dos fármacos , Proteínas Recombinantes de Fusão/imunologia , Cloreto de Sódio/farmacologia , Linfócitos T Citotóxicos/imunologia , Proteínas do Core Viral/imunologia , Proteínas Virais de Fusão/imunologiaRESUMO
Sequential poly(Arg-Thr-Lys-Pro) consisting mainly of the repeat of tuftsin Thr-Lys-Pro-Arg was synthesized by condensing the p-nitrophenyl ester of Arg(HCl)-Thr-Lys-(2-Cl-Z)-Pro in the presence of HOBt. Two haptenic sequences of the Pre-S region of hepatitis B virus antigen (10-26 and 39-55) were prepared by solid phase and coupled to polytuftsin via glutaraldehyde. The peptides, either free or coupled to polytuftsin, were administrated to mice and the antisera were assayed by ELISA. Coupling the peptides to the polypeptide significantly improved the anti-peptide antibody titer in Freund complete adjuvant or in NaCl 0.9%. Cross-reaction between antibodies induced by the peptides and the native protein was also improved. Polytuftsin alone is very poorly immunogenic.
Assuntos
Antígenos de Superfície da Hepatite B/imunologia , Polímeros/síntese química , Tuftsina/síntese química , Animais , Cromatografia Líquida de Alta Pressão/métodos , Portadores de Fármacos , Ensaio de Imunoadsorção Enzimática , Soros Imunes , Indicadores e Reagentes , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Polímeros/imunologia , Tuftsina/imunologiaRESUMO
This paper describes the single channel properties of a series of synthetic analogues of gramicidin A, where all four tryptophans are replaced either by tyrosine or by several O-protected (benzyl, methyl, ethyl or t-butyl) derivatives. It is shown that, although all analogues bear similar dipole moment on their side-chains, the conductance depends on the hydrophobicity of these protecting groups. An analysis of the conductance data suggests that the conductance is governed by the binding process and a possible explanation, based on conformational considerations, is proposed.
Assuntos
Gramicidina/química , Tirosina/química , Sequência de Aminoácidos , Condutividade Elétrica , Canais Iônicos/química , Canais Iônicos/fisiologia , Lipídeos de Membrana/química , Lipídeos de Membrana/fisiologia , Membranas/química , Membranas/fisiologia , Dados de Sequência Molecular , Relação Estrutura-Atividade , Triptofano/químicaRESUMO
The behavior of four linear gramicidins, which differ by the nature of their 9, 11, 13, and 15 aromatic residues, together with a covalent "head to tail" retro GA-DAla-GA dimer, has been examined at the air-water interface. It is shown that all four "monomers" have almost the same molecular area, which is compatible with either a single-stranded or a double-stranded helical model, whereas it is suggested that retro GA-DAla-GA could adopt another conformation. The surface potential measurements agree with those of different groups of molecules characterized by their single-channel behaviors.
Assuntos
Gramicidina , Canais Iônicos/fisiologia , Modelos Biológicos , Pressão , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
An amphipathic polypeptide, Pn, with a tandemly repeated LKELPEKL sequence including a proline every eight residues, as well as a series of shorter peptides having the same sequence, P2, P3, P4, P5 and P6, were synthesized. Their conformation in aqueous solution was mainly studied by CD. At low temperature, these peptides and polypeptides are completely unordered and undergo a reversible transition leading to a partly alpha-helical structure upon heating. Such behavior has been demonstrated for a few proteins by other authors and has been called cold-denaturation. The transition temperature of the polypeptide is close to 20 degrees C. The conformational change does not depend on concentration, indicating a monomolecular process. The high-temperature structure seems to be compact as for globular proteins. A model of folded structure is proposed from experimental data and from molecular modelling studies.