Marius Ptak

A NMR study of the ionization of fatty acids, fatty amines and N-acylamino acids incorporated in phosphatidylcholine vesicles

The ionization of fatty acids, fatty amines and N-acylamino acids incorporated in phosphatidylcholine single-walled vesicles has been measured. The guest molecules have been specifically enriched with 13C and titrated by using NMR spectroscopy. The apparent pKa of fatty acids in phosphatidylcholine bilayers if 7.2-7.4 and those of fatty amines are approx. 9.5. These pKa values depend on many different parameters related to the structure of the lipid/solution interface, to the composition of the aqueous medium and to the localization of the ionizable groups. A special sensitivity to the ionic strength and to the surface charge has been found. A positive surface charge decreases the pKa value whereas a negative one increases it, the total range of variation being 2.5-3 units. In a qualitative macroscopic interpretation, it is proposed that pKa is essentially determined by the low polarity of the lipidic matrix.

Surfactin/iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturin A.

Iturin A and surfactin are two lipopeptides extracted from a same strain of Bacillus subtilis. Iturin A possesses antibiotic and antifungal activities and surfactin is a strong surfactant. The presence of surfactin, at a concentration at which, alone, it is inactive, increases to a very large extent the haemolysis percent induced by iturin A. This synergistic effect seems to be in relation with interactions between iturin A and surfactin. Iturin A adsorbs to and penetrates into surfactin monolayers. Iturin A and surfactin are miscible and interact specifically in mixed monolayers.

Two-dimensional 1H NMR study of recombinant insect defensin A in water: resonance assignments, secondary structure and global folding.

SummaryA 500 MHz 2D1H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities,3JNH-αH coupling constants as well as1H/2H exchange rates and Δδ/ΔT temperature coefficients of NH protons strongly support the existence of an α-helix (residues 14–24) and of an antiparallel β-sheet (residues 27–40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from3JNH-αH coupling constants, (iii) 12 hydrogen bonds mostly deduced from1H/2H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a β-sheet is linked to an α-helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.

Interfacial properties of surfactin

Surfactin is a lipopeptide produced by various strains of Bacillus subtilis and is a very powerful surfactant. Here we present the first report on the interfacial behavior of surfactin. The adsorption, Γmax, of surfactin at the interface of diluted solutions (5 × 10−8 to 5 × 10−7M) is around 3 × 1018 molecule m−2, a value indicating that surfactin molecules are in a very packed situation. Surfactin spreads readily at the airwater interface: the equilibrium spreading pressure πe ≈ 30 mN m−1 reaches 45 mN m−1 when electrolytes (KCl or CaCl2) are dissolved in the alkaline subphase. We have plotted the compression isotherm curves and determined surface parameters. These parameters vary only a little with temperature but are very affected by the pH of the subphase. Plotting the transition pressure value πt as a function of pH results in a titration curve from which one can deduce the pK value of surfactin at the interface. This value, pKs ≈ 6 is around 2 pH units higher than the pK of surfactin in solution. The addition of electrolytes (I = 0.15 M) in the alkaline subphase leads to the neutralization of the surfactin monolayer (protonation of the acidic residues LGlu1 and LAsp5 of the peptide cycle). This neutralization is complete in the case of Ca++ ions but not in the case of monovalent cations (Na+ or K+). When surfactin monolayers are subjected to successive compression—expansion cycles we observe a reproducible hysteresis loop. The surface parameters of surfactin are compared to those of iturins, lipopeptides also extracted from B. subtilis, the structure and properties of which are very similar.

Pore-forming properties of iturin A, a lipopeptide antibiotic

The addition of iturin A, a lipopeptide antibiotic extracted from Bacillus subtilis, to a bimolecular lipid membrane (BLM) increases dramatically its electrical conductance. For very low concentration of iturin A, discrete conductance steps are observed which are assigned to the formation of conducting pores. The characteristics of these pores depend on the lipid content of the BLM and they change with time. Cholesterol considerably increases the lifetimes of open states. The pores are slightly anion versus cation selective. These first observations unable us to briefly discuss the pore-forming properties of lipopeptides.

Ionization of fatty acids at the lipid—water interface

As demonstrated for phosphatidic acid [1 ] and for phosphatidyl serine [2], thermodynamic stability of lipid phases strongly depends on the ionization of the polar heads. Interfacial ionization phenomena must also be considered in interactions between ionizable extrinsic molecules and phospholipids as they could interfere in membrane stability and membrane function, especially in proton and ion transport. Fluorescent pH indicators have been used [3] to determine interfacial ionization. We use fatty acids as ionizable probes to investigate the interface in which the carboxylic group is anchored. For electron spin resonance (ESR) experiments, stearic acid was spinlabelled on the C5 position. For nuclear magnetic resonance (NMR) experiments, stearic and palmitic acids were selectively enriched with 13C on the carboxylic group. These probes were incorporated in egg lecithin vesicles or multilayers, in which the zwitterionic phosphatidyl choline head groups define a neutral host system for the carboxylic group.

1H NMR and fluorescence studies of the complexation of DMPG by wheat non‐specific lipid transfer protein. Global fold of the complex

Plant non‐specific lipid transfer proteins (LTPs) are proteins which transfer lipids between membranes in vitro and are believed to be involved in the transport of cutin monomers to the cuticle layer in vivo or in the plant defence against phytopathogens. The complexation of DMPG, a diacyl phospholipid, by wheat ns‐LTP, a protein extracted from wheat seeds, was followed by 1H NMR and fluorescence spectroscopy. The global fold of the protein was calculated using the DIANA software package from a list of 968 distance constraints. The internal cavity volume, a feature common to all known ns‐LTP structures, was estimated to be 750 Å3 using the ‘CAVITE’ program. This model of the complex was obtained by inserting a lipid molecule in the cavity and was energy minimized. The study showed that the protein fold described for the free form was only weakly affected by the insertion of the bulky lipid. Observation of some intermolecular NOEs between the protein and the lipid glycerol moiety revealed that the cavity entrance was located between residues His35 and Arg44. The resulting solution structure was compared to the crystal structure of the maize ns‐LTP/palmitate complex.

Production, isolation and characterization of (Leu4) - and (Ile4)surfactins from Bacillus subtilis

Bacillus subtilis coproduces several surfactin variants that are powerful biosurfactants and have potential applications in biology and industry. A single amino acid substitution in the heptapeptide moiety of surfactins strongly modifies their properties. To better establish structure-activity relationships and to search new variants with enhanced properties, Bacillus subtilis was grown into two modified culture media. Two new variants were isolated by chromatographic methods and studied by NMR spectroscopy. As planned, modifications consisted in the substitution of the l-valine residue at the fourth position by a more hydrophobic residue, i.e., leucine or isoleucine. These [Leu4]- and [Ile4]surfactins have a higher affinity for hydrophobic solvents and a twice improved surfactant power. Structure-property correlations were confirmed by analysis of the hydrophobic residue distribution in the three-dimensional model of the structure of surfactin in solution.

Lipopeptides with Improved Properties: Structure by NMR, Purification by HPLC and Structure–Activity Relationships of New Isoleucyl‐rich Surfactins

The biosynthesis of bacterial isoleucyl‐rich surfactins was controlled by supplementation of L‐isoleucine to the culture medium. Two new variants, the [Ile4,7]‐ and [Ile2,4,7]surfactins, were thus produced by Bacillus subtilis and their separation was achieved by reverse‐phase HPLC. Amino acids of the heptapeptide moiety were analysed by chemical methods, and the lipid moiety was identified to β‐hydroxy anteiso pentadecanoic acid by combined GC/MS. Sequences were established on the basis of two‐dimensional NMR data. Because conformational parameters issuing from NMR spectra suggested that the cyclic backbone fold was globally conserved in the new variants, structure–activity relationships were discussed in details on the basis of the three‐dimensional model of surfactin in solution. Indeed, both variants have increased surface properties compared with that of surfactin, and this improvement is assigned to an increase of the hydrophobicity of the apolar domain favouring micellization. Furthermore, the additional Leu‐to‐Ile substitution at position 2 in the [Ile2,4,7]surfactin leads to a substantial increase of its affinity for calcium, when compared with that of [Ile4,7]surfactin or surfactin. This effect is assigned, from the model, to an increase in the accessibility of the acidic side chains constituting the calcium binding site. Thus, the propensities of such active lipopeptides for both hydrophobic and electrostatic interactions were improved, further substantiating that they can be rationally designed.

Comparison of solution and crystal structures of maize nonspecific lipid transfer protein: a model for a potential in vivo lipid carrier protein.

The three‐dimensional solution structure of maize nonspecific lipid transfer protein (nsLTP) obtained by nuclear magnetic resonance (NMR) is compared to the X‐ray structure. Although both structures are very similar, some local structural differences are observed in the first and the fourth helices and in several side‐chain conformations. These discrepancies arise partly from intermolecular contacts in the crystal lattice. The main characteristic of nsLTP structures is the presence of an internal hydrophobic cavity whose volume was found to vary from 237 to 513 Å3 without major variations in the 15 solution structures. Comparison of crystal and NMR structures shows the existence of another small hollow at the periphery of the protein containing a water molecule in the X‐ray structure, which could play an important structural role. A model of the complexed form of maize nsLTP by α‐lysopalmitoylphosphatidylcholine was built by docking the lipid inside the protein cavity of the NMR structure. The main structural feature is a hydrogen bond found also in the X‐ray structure of the complex maize nsLTP/palmitate between the hydroxyl of Tyr81 and the carbonyl of the lipid. Comparison of 12 primary sequences of nsLTPs emphasizes that all residues delineating the cavities calculated on solution and X‐ray structures are conserved, which suggests that this large cavity is a common feature of all compared plant nsLTPs. Furthermore several conserved basic residues seem to be involved in the stabilization of the protein architecture. Proteins 31:160–171, 1998.