Influence of the conformation of a macromolecule on the generation of T-cell proliferative response. A study with model polypeptides.

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.

Experimental and theoretical study of gramicidin P, an analog of gramicidin A with a methylamine C-terminal.

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.

Efficient binding to the MHC class I K(d) molecule of synthetic peptides in which the anchoring position 2 does not fit the consensus motif.

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.

Single channels and surface potential of linear gramicidins.

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.

Conformations of gramicidin A and its 9,11,13,15-phenylalanyl analog in dimethyl sulfoxide and chloroform.

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.

Analysis of the ion transfer through the channel of 9,11,13,15-phenylalanylgramicidin A.

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.

Synthesis, conformation and reactivity towards p-nitrophenyl acetate of polypeptides incorporating aspartic acid, serine and histidine.

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.

Synthesis of the sequential polypeptide poly[Cys(Acm)-Ser-Phe-Glu-Glu] as a model for hydrolytic enzymes.

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).

Reactivity of sulfhydryl group in peptides and poly-peptides with p-nitrophenylacetate. S to N acyl shift in cysteine.

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".

Synthesis and characterization of Tyr(Bzl)9,11,13,15 and Tyr9,11,13,15 gramicidin A.

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).

Studies of the influence of different cross-linking reagents on the immune response against a B-epitope.

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.

Purification of synthetic peptide libraries by affinity chromatography using the avidin-biotin system.

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.

Single channels of 9, 11, 13, 15-destryptophyl-phenylalanyl-gramicidin A.

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.

Mixed monolayers of linear gramicidins and phospholipid. Surface pressure and surface potential studies.

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.

Synthesis and interfacial behavior of poly(Lys-Tyr-Tyr-Lys).

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.

Structural features of a chimeric peptide inducing cytotoxic T lymphocyte responses in saline.

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.

Synthesis of a new carrier for immunization: polytuftsin. Two examples of its use with peptides selected in the hepatitis B surface antigen.

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.

High thermal stability and cold-denaturation of an artificial polypeptide.

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.

Use of synthetic peptide libraries for the H-2Kd binding motif identification.

To identify Kd-binding peptides, an approach based on small peptide libraries has been developed. These peptide libraries correspond to all possible single-amino acid variants of a particular Kd-binding peptide, SYIPSAEYI, an analog of the Plasmodium berghei 252-260 antigenic peptide SYIPSAEKI. In the parent sequence, each position is replaced by all the genetically encoded amino acids (except cysteine). The multiple analog syntheses are performed either by the Divide Couple and Recombine method or by the Single Resin method and generate mixtures containing 19 peptides. The present report deals with the synthesis, the purification, the chemical characterization by amino acid analysis and electrospray mass spectrometry (ES-MS), and the application of such mixtures in binding tests with a soluble, functionally empty, single-chain H-2Kd molecule denoted SC-Kd. For each mixture, bound peptides were eluted and analyzed by sequencing. Since the binding tests were realized in noncompetitive conditions, our results show that a much broader set of peptides bind to Kd than expected from previous studies. This may be of practical importance when looking for low affinity peptides such as tumor peptides capable of eliciting protective immune response.

Interactions of basic amphiphilic peptides with dimyristoylphosphatidylcholine small unilamellar vesicles: optical, NMR, and electron microscopy studies and conformational calculations.

The interactions of DMPC small unilamellar vesicles with four amphiphilic polypeptides [(LKKL)n, (LRRL)n, (LKKL)4, and (YKKY)n] have been investigated by circular and infrared dichroism, turbidimetry, electron microscopy, and fluorescence, 1H, and 31P nuclear magnetic resonance spectroscopy. The main results obtained are the following: (1) Well-defined complexes are formed by the association of one amino acid residue with approximately two lipid molecules. (2) In the presence of polypeptides fusions are observed between SUVs when the molar ratio p is less than 0.05, and a clearance effect is observed when p is higher than 0.05. (3) The anchoring sites of the polypeptides on DMPC molecules are the negative phosphate groups through electrostatic interactions with the terminal NH3+ of lysine residues. (4) The polypeptides adopt an alpha-helical conformation with their axis parallel to the membrane surface. The hydrophobic part of the amphiphilic alpha helix can penetrate the outer lipid leaflet down to the C5 position. (5) Choline methyl groups are not involved in the interactions between lipid molecules and amino acid residues. (6) Phosphorus atom mobility around the P-O-glycerol bond is strongly reduced whereas that of methylene groups is progressively weakened when going up from C13 to C1. Finally, using modeling and energy calculations a model of possible Ac(LKKL)4NHEt-DMPC SUV complexes is presented.