Conformation study of HA(306-318) antigenic peptide of the haemagglutinin influenza virus protein.

Several HLA-DR alleles present the immunodominant HA(306-318) peptide of haemagglutinin of the influenza virus to T cells. NMR data of the peptide in various water solutions exclude any alpha-helix or turn conformations. Circular dichroism and Fourier transform infrared spectroscopies indicate an estimated beta-extended structure in water of 31% and 28%, respectively, with spectra shape similar to the ones observed for beta-sheet containing proteins. The H/D amide exchange suggests a stable length-dependent interchain hydrogen-bonding. The partially beta-extended conformation of HA(306-318) in solution might be close to the one found in HA(306-318)-HLA-DR1 complex. These results suggest different interconverting extended conformations of HA(306-318), depending on the microenvironment of the solution medium. This flexibility emphasizes the ability of some peptides to fit more easily the binding site of several HLA-DR molecules. Similar results were obtained on the HIV P25(263-277) peptide which has been previously shown to be a good DR1 binder. From a vibrational point of view, infrared Amide I frequencies of secondary structures in peptides were ascertained. As previously demonstrated for proteins in solution, Fourier transform infrared and circular dichroism spectroscopies appear to be valuable tools for conformational properties of peptides. Their use may contribute to the detection of peptide conformation-binding relationship which has to be further tested by biochemical and biological studies.

Does phosphoryl protonation occurs in aqueous phosphoesters solutions.

Ionisation of trimethylphosphate (TMP), dimethylphosphate (DMP) and diethylphosphate (DEP) is investigated by acidic titration in water by Raman (R), Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies. The vibrational frequencies of the PO(2)(-) ionic form and the neutral form were found in accord with the literature. While increasing further H(+) concentration, the PO band disappears in the benefit of new ones. These results, together with deuteration experiments indicate the presence of a new ionic form positively charged with general formula R(1)R(2)R(3)P(OH)(+) or R(1)R(2)P(OH)(+)(2). The pK of this phosphonium entities is lying in the range -2, -4. These results were confirmed by (31)P NMR titration. The occurrence of such a phosphonium ion in aqueous solutions might be of crucial importance for biochemical reactions and interactions, owing to the large spread of phosphoryl group in biomolecules and keeping in mind that intracellular compartments are more likely concentrated media with little free water than real aqueous solutions. Furthermore, pK's can be shifted by physical-chemical parameters like dielectric constant and electric field. This may involve at least fractional positive charge apparition that might be important in biochemical regulation by charge-charge and charge-dipole interactions. This finding will gain to be further explored on more complex molecules like phospholipids, nucleic acids and proteins.

Residue 67 in the DRbeta1*0101 and DRbeta1*0103 chains strongly influences antigen presentation and DR-peptide molecular complex conformation.

Two closely-related molecules, DR(alpha,beta1*0101) and DR(alpha,beta1*0103), whose beta chains only differ by three amino acids at positions 67, 70, and 71, and six intermediate molecules obtained by site-directed mutagenesis were used to ascertain the respective roles of the three polymorphic residues. Substitutions at positions 70 (D-->Q), 71 (E-->R) and 67 (I or L-->F) strongly affected HA 306-318-specific T-cell recognition. The consequences of the substitution of residue 67 by a phenylalanine depended on the modified HLA-DR molecule. Although this substitution completely inhibited peptide-specific DR1-restricted T-cell recognition, its manifestations on the DR103-restricted T-cell response were variable (abolishing proliferation of some cell lines and not others), no matter what the peptide presented was (HA 306-319 or HIV P25 peptides). We also observed that inhibition of the proliferation of an alloreactive anti-DR103 T-cell clone, caused by a substitution at position 70, was completely cancelled by substitution of residue 67 by a phenylalanine. The observations based on functional experiments, thus, suggest that residue 67 plays an important role in determining conformation of the peptide presented to the T cells. Molecular modeling was used to predict changes induced by amino acid substitutions and highly supports functional data. Substitution of residue 67 by a phenylalanine could have repercussions on the structure of HLA-DR molecule/peptide complexes and affect T-cell recognition.

Role of polymorphic residues of human leucocyte antigen-DR molecules on the binding of human immunodeficiency virus peptides.

A study was made of the binding properties of 96 human immunodeficiency virus peptides to human leucocyte antigen (HLA)-DR1 and HLA-DR103 molecules, which differ by three amino acids at positions 67, 70 and 71 in the beta chains. The affinity of the peptides was characterized by their inhibitory concentrations in competitive binding assays which displace half of the labelled influenza haemagglutinin peptide HA306-318 (IC50). Among the high-affinity peptides (IC50 < or = 1 microM), seven bound to DR1, three to DR103 and five equally well to both alleles (promiscuous peptides). Thirty-two other peptides showed medium or low affinity for DR molecules. The role of polymorphic residues was analysed using six mutated DR molecules, intermediates between DR1 and DR103 and differing by one or two substitutions at positions 67, 70 or 71. We reached the same conclusions when using DR1-specific or DR103-specific peptides: modification of residue 70 had no effect on peptide affinity, but single substitution at positions 67 or 71 decreased the allele specificity of the peptides while double substitution at 67 and 71 completely reversed the peptide specificity. In functional assays, DR-binding peptides are able to outcompete specific T-cell proliferation. Furthermore, modification at position 67 or 70 significantly affects the T-cell response and mutation at position 71 abolishes completely the T-cell proliferation. Thus, the polymorphic positions 67 and 71 contributed to the peptide binding with direct effects on T-cell receptor (TCR) recognition while position 70 seems to be mostly engaged in TCR interactions. Furthermore, our results suggest that polymorphic residues may select allele-specific peptides and also influence the conformation of promiscuous peptides.

Interactions of mycobacterial glycopeptidolipids with membranes: influence of carbohydrate on induced alterations.

Glycopeptidolipids (GPLs) are specific constituents of mycobacteria known as opportunistic pathogens. The influence of the carbohydrate moiety on GPL-induced membrane alterations was examined with GPLs bearing 1-5 sugar residues (GPL-1 to GPL-5) and a sulfated GPL (S-GPL-2). GPLs decreased the ADP/O ratio and increased controlled respiration of isolated mitochondria. The more polar GPLs were the less active, with the following order of efficiency: GPL-1 > GPL-2 > S-GPL-2 = GPL-3 = GPL-5. GPL-1 and GPL-2 increased passive permeability of liposomes to carboxyfluorescein (GPL-1 > GPL-2), while GPL-3 and GPL-5 were inactive. GPL-2 and GPL-3 decreased the transmembrane electrical potential (delta psi) in isolated mitochondria (GPL-2 > GPL-3). These results suggest that GPLs uncouple oxidative phosphorylation by increasing the passive permeability of the mitochondrial membrane to protons. Compression isotherms of GPL-2 monolayers showed that, at low surface pressure, the area per GPL-2 molecule was about 5 times that of an acyl chain: it is likely that the peptide moiety was at the air/water interface. With an increase in the surface pressure, its area decreased, down to that of a tightly packed acyl chain. It is postulated that the glycopeptidic moiety can be either at in the interface or dipping into the water.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between trypanocidal drugs and membrane phospholipids. A surface pressure, surface potential and electrophoretic mobility study.

Amphiphilic diphenyl methane derivatives exhibiting both antiproliferative and trypanocidal effects were studied with respect to their interactions with phospholipids, in monolayers and bilayers. These compounds, namely (4-benzyl)-phenoxy-2 trimethylammonium ethane iodide (D1), (4-tertiobutyl)-phenoxy-2 morpholinium ethane chloride (D2), and (4-benzyl)-phenoxy-2 morpholinium ethane chloride (D3), were shown to interact with phosphatidylcholine (PC) and phosphatidylserine (PS) in monolayers, as monitored by surface pressure and surface potential measurements. The film expansion of monolayers, on 10 mM NaCl subphase at pH 7.1, was more pronounced in the presence of D2 and D3 in the subphase before spreading of the lipids than with the injection of the drugs underneath a preformed film. Apparent binding constants of 10(4) M-1 were determined for both drugs from monolayer experiments. With D2 in the presence of PS, results of monolayer compressions and electrophoretic mobility measurements indicate binding of the drug to the lipid molecules only when the molecular area was large. D3 was shown to interact with PS, both in monolayers and bilayers, with a drug-to-lipid binding constant of about 2 x 10(4)M-1, as evaluated from electrophoretic mobility measurements on PS liposomes. These results, which indicate binding of these drugs to phospholipids in the order D2 less than D3, correlate with the biological activity of the drugs, and may account for the discrepancy observed between the drug concentrations required for biological and binding activities.

Mycobacteria glycolipids as potential pathogenicity effectors: alteration of model and natural membranes.

Four mycobacterial wall glycolipids were tested for their effects on phospholipidic liposome organization and passive permeability and on oxidative phosphorylation of isolated mitochondria. From fluorescence polarization of diphenylhexatriene performed on liposomes it was concluded that the two trehalose derivatives (dimycoloyltrehalose and polyphthienoyltrehalose) rigidified the fluid state of liposomes, the triglycosyl phenolphthiocerol slightly fluidized the gel state, while the peptidoglycolipid ("apolar" mycoside C) just shifted the phase transition temperature upward. Dimycoloyltrehalose was without effect on liposome passive permeability, as estimated from dicarboxyfluorescein leak rates, and polyphthienoyltrehalose and triglycosyl phenolphthiocerol slightly decreased leaks, while mycoside C dramatically increased leaks. Activity of these lipids on mitochondrial oxidative phosphorylation was examined. The two trehalose derivatives have been tested previously: both had the same type of inhibitory activity, dimycoloyltrehalose being the most active. Triglycosyl phenolphthiocerol was inactive. Mycoside C was very active, with effects resembling those of classical uncouplers: this suggested that its activity on mitochondria was related to its effect on permeability. All these membrane alterations were called nonspecific because it is likely that they result from nonspecific lipid-lipid interactions, and not from recognition between specific molecular structures. Such nonspecific interactions could be at the origin of some of the effects of mycobacteria glycolipids on cells of the immune system observed in the last few years.

Repeat peptide motifs which contain beta-turns and modulate DNA condensation in chromatin.

In order to understand better the roles of repeating basic peptide motifs in modifying DNA structure, we have synthesized typical repeats found in the C-terminal domain of histone H1 (KTPKKAKKP)2 and in the N-terminal domain of nucleolin (ATPAKKAA)2. By using circular dichroism in conjunction with Raman and Fourier-transform infrared spectroscopies, we demonstrate that the abilities of the two peptides to affect DNA conformation are dramatically different. Whilst the binding of the nucleolin repeat to DNA does not significantly alter its conformation, the binding of H1 repeat induces a very marked DNA condensation, giving rise to a psi(-)-type circular dichroic spectrum. The H1 repeat thus adopts a more rigid beta-turn-containing structure which probably binds to the DNA minor groove as assessed by competition with the drug Hoechst 33258. Unexpectedly, the DNA condensation induced by the H1 repeat is enhanced by the nucleolin repeat which by itself does not promote any alteration in DNA conformation.

Effect of pH and monovalent cations on the ionization state of phosphatidylglycerol in monolayers. An experimental (surface potential) and theoretical (Gouy-Chapman) approach.

Ionization of the acidic phospholipid phosphatidylglycerol has been studied by measuring the surface potential of monomolecular films of the lipid as a function of the aqueous subphase pH and the concentration of monovalent cations (Li, Na, Cs). It is shown that the experimental data can be interpreted by means of the Gouy-Chapman theory in its simplest formulation, provided an adsorption of cations at the membrane surface is accounted for. This allows us to predict the ionization state of the lipid for given ionic conditions in the subphase. Above pH 4, for subphase ion concentration higher than 10 mM, or for ion concentrations above 0.1 mM at pH 5.6, phosphatidylglycerol is fully deprotonated. Within the limits of our theoretical approach, association constants of the cations to the lipid lie around 0.1-0.6 M-1.

Effect of pH, mono- and divalent cations on the mixing of phosphatidylglycerol with phosphatidylcholine. A monolayer (pi, delta V) and fluorescence study.

The mixing of various molecular species of phosphatidylglycerol and phosphatidylcholine differing in their acyl chain lengths has been studied both in monolayers (pi, delta V), and in water dispersions (fluorescence polarization) with varying pH and ionic strength of the aqueous phase and in the presence of the divalent cations Mg2+ and Ca2+. In dilauroylphosphatidylglycerol/dipalmitoylphosphatidylcholine mixtures, both in monolayers and in water dispersions, no phase separation was detected at pH 2.9 where phosphatidylglycerol was protonated. With dipalmitoylphosphatidylglycerol/dipalmitoylphosphatidylcholine mixtures, in monolayers and at the same pH, no phase separation was detected for surface pressures below pi = 40 mN.m-1. In monolayers, and under ionic conditions such that phosphatidylglycerol was ionized (pH 5.6, 10 mM NaCl) miscibility was observed with dilauroylphosphatidylglycerol and dipalmitoylphosphatidylcholine and also with dipalmitoylphosphatidylglycerol and dilauroylphosphatidylcholine. Varying the ionic strength did not alter the miscibility of these lipids. The divalent cations Mg2+ and Ca2+ did not modify that of dilauroylphosphatidylglycerol with dilauroylphosphatidylcholine or with dipalmitoylphosphatidylcholine. Both in monolayers and in water dispersions, dipalmitoylphosphatidylglycerol and dilauroylphosphatidylcholine appeared to be at least partly miscible, in the presence of magnesium. Only in the presence of calcium and at high surface pressure might the monolayer data account for phase separation between these two lipids. The data presented demonstrate the existence of strong cohesive forces between phosphatidylcholine and phosphatidylglycerol with a marked influence of the former on the physical state of the latter. From an analysis of the delta V data, it is suggested that intrafacial hydrogen bonds may play a significant role in stabilizing phosphatidylcholine/phosphatidylglycerol mixtures.

Phase behaviour in monolayers and in water dispersions of mixtures of dimannosyl diacylglycerol with phosphatidylglycerol.

Mixtures of dimannosyl diacylglycerol, extracted from the membrane of Micrococcus luteus, with synthetic dipalmitoyl phosphatidylglycerol or with samples of phosphatidylglycerol and phosphatidylinositol, extracted from the same bacterium, have been studied. Through a monolayer (pi, delta V) study and from fluorescence polarization data relative to diphenylhexatriene embedded in vesicles of the mixed lipids, it is shown that the glycolipid interacts with the phospholipids. These interactions are independent of the structure and physical state of the phospholipid acyl chains, of the lipid molecular packing and of the nature of the cations (monovalent, bivalent) present in the aqueous phase. No phase separation was detected, either in monolayers or in water dispersions. Furthermore, the data presented demonstrated a marked influence of the glycolipid on the phase behaviour of phosphatidylglycerol, both in the presence of monovalent (Na+, K+) and bivalent (Ca2+, Mg2+) cations. This point is of particular interest with regard to the highly rigid phase this phospholipid is known to assume in the presence of bivalent cations. It is then suggested that the glycolipid could act as a regulator of the membrane fluidity by preventing a too high rigidity of the lipid phase when bivalent cations are present at the membrane surface.