Interaction of mastoparan with membranes studied by 1H-NMR spectroscopy in detergent micelles and by solid-state 2H-NMR and 15N-NMR spectroscopy in oriented lipid bilayers.

Several complementary NMR approaches were used to study the interaction of mastoparan, a 14-residue peptide toxin from wasp venom, with lipid membranes. First, the 3D structure of mastoparan was determined using 1H-NMR spectroscopy in perdeuterated (SDS-d25) micelles. NOESY experiments and distance geometry calculations yielded a straight amphiphilic alpha-helix with high-order parameters, and the chemical shifts of the amide protons showed a characteristic periodicity of 3-4 residues. Secondly, solid-state 2H-NMR spectoscopy was used to describe the binding of mastoparan to lipid bilayers, composed of headgroup-deuterated dimyristoylglycerophosphocholine (DMPC-d4) and dimyristoylphosphatidylglycerol (DMPG). By correlating the deuterium quadrupole splittings of the alpha-segments and beta-segments, it was possible to differentiate the electrostatically induced structural response of the choline headgroup from dynamic effects induced by the peptide. A partial phase separation was observed, leading to a DMPG-rich phase and a DMPG-depleted phase, each containing some mastoparan. Finally, the insertion and orientation of a specifically 15N-labeled mastoparan (at position Ala10) in the bilayer environment was investigated by solid-state 15N-NMR spectroscopy, using macroscopically oriented samples. Two distinct orientational states were observed for the mastoparan helix, namely an in-plane and a trans-membrane alignment. The two populations of 90% in-plane and 10% trans-membrane helices are characterized by a mosaic spread of +/- 30 degrees and +/- 10 degrees, respectively. The biological activity of mastoparan is discussed in terms of a pore-forming model, as the peptide is known to be able to induce nonlamellar phases and facilitate a flip-flop between the monolayers.     

Distribution of alginate oligomers in Catharanthus roseus and Wasabia japonica cells cultured in a medium containing alginate oligomers

Distribution of alginate oligomers (AO) which are endogenous elicitor-like substances, in cultured plant cells were investigated by using AO conjugated with monopotassium 7-amino-1,3-naphthalenedisulfonate (ANDS). When AO-ANDS was added at 0.5 g l^–1 to the Catharanthus roseus cell culture, it adhered to the cells as observed by fluorescence microscopy. Using protoplasts of C. roseus, AO-ANDS was found not only in the cell walls but also in the cell membrane and cytoplasm. When C. roseus was cultivated in a medium containing oligo-galacturonic acids, as an endogenous elicitor, this was also found in the cell wall, cell membrane and cytoplasm of C. roseus cells. Similar results were also obtained with Wasabia japonica cells.     

A fibronectin-binding protein from rice bran with cell adhesion activity for animal tumor cells.

A rice bran 57-kDa protein was isolated by affinity chromatography with fibronectin immobilized on agarose. This fibronectin-binding protein designated as RB-57 had an amino-terminal amino acid sequence identical with that of a putative mature form of rice hydroxyproline-rich glycoprotein. A distinct feature of the amino acid composition of RB-57 was the high contents of hydroxyproline and proline representing about 45% of the total amino acids. The sugar analysis indicated that arabinose represented 46.8% of the total carbohydrates. RB-57 showed cell adhesion activity for murine Lewis lung carcinoma cells. The result suggests that RB-57 may play a role in plant cell adhesion, although cell adhesion-promoting activity for plant cells remains to be tested.     

Carbohydrate recognition of gramicidin S analogues in aqueous medium.

We have designed and synthesized of carbohydrate-binding peptides, gramicidin S analogues. Asn/Asp/Gln and Trp residues in the peptides were employed as the binding sites for carbohydrates by hydrogen-bonding interaction and the creation units for hydrophobic pocket to promote the interaction, respectively. The data of fluorescence spectroscopy and affinity column chromatography indicated that the peptides possessed the binding ability for some carbohydrates in aqueous medium. As a result of 1H NMR study, nuclear Overhauser effects between aromatic side chains of a peptide, [Gln(1,1'),Trp(3,3')]-gramisidin S and mannose were observed, indicating that the interaction of the peptide with the sugar occurred in the hydrophobic environment formed by Trp and Phe residues.     

Conformational behavior of oxygenated mycobacterial mycolic acids from Mycobacterium bovis BCG

Phase diagrams of Langmuir monolayers of oxygenated mycolic acids, i.e. methoxy mycolic acid (MeO-MA), ketomycolic acid (Keto-MA), and artificially obtained deoxo-mycolic acid (deoxo-MA) from Mycobacterium bovis BCG were obtained by thermodynamic analysis of the surface pressure (pi) vs. average molecular area (A) isotherms. At lower temperatures and lower surface pressures, both Keto- and MeO-MAs formed rigid condensed monolayers where each MA molecule was considered to be in a 4-chain form, in which the three carbon chain segments due to bending of the 3-hydroxy aliphatic carboxylate chain and the 2-side chain were in compact parallel arrangement. At higher temperatures and surface pressures, MeO-MA and deoxo-MA tended to take stretched-out conformations in which the 3-hydroxy aliphatic carboxylate chain was more or less in an extended form, but Keto-MA retained the original 4-chain structure. The thickness measurement of the monolayers in situ by ellipsometry at different pi values and temperatures supported the above conclusions derived from the phase diagrams. The enthalpy changes associated with the phase transitions of MeO-MA and deoxo-MA implied that the MeO-MA needed larger energy to change from a compact conformation to an extended one, possibly and partly due to the dehydration of the methoxy group from water surface involved. Molecular dynamics studies of MA models derived from Monte Carlo calculations were also performed, which confirmed the conformational behavior of MAs suggested by the thermodynamic studies on the Langmuir monolayers.