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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.

The structure of [Arg8]vasopressin methylenedithioether ([AVP]CH2) has been determined in dimethylsulfoxide-d6. Two-dimensional DQF-COSY and NOESY spectra were measured and used to derive angle and distance constraints for restrained molecular dynamics (MD) calculations. In the MD trajectory, two types of beta-turn structure were found in the region from Tyr2 to Asn5, suggesting an equilibrium between type-I and type-II' beta-turn structures. When Halpha chemical shifts were used as an additional constraint, the type-I turn was favoured. To validate this result, an independent energy minimization procedure was used, using differences between calculated and observed chemical shifts. The two approaches gave essentially identical results. It is therefore concluded that the type-I turn predominates in solution. Analysis of calculated chemical shift contributions suggests that the beta-turn structure found in AVP is well preserved in [AVP]CH2, although the pressin ring size is expanded.

The structure of the bee venom peptide melittin has been determined in water at pH 6.0, 50 mM sodium phosphate, at various temperatures. At all temperatures studied, the peptide is tetrameric, and consists of two helical regions (residues 2-11 and 13-23) with an unstructured C-terminal region. The connection between the helices (residues 11-13) is well defined. At 30 degrees C, the structure of the monomeric unit has been characterised using NOEs, and a family of structures is presented (root-mean-square deviation to the mean structure 1.4 A over the structured residues), with low NOE violations and good stereochemistry. The angle between the helices is 46+/-13 degrees, and the structure is very similar to the previously determined crystal structure of the aqueous tetramer. The peptide forms a tetramer that is made up from a dimer of dimers. The structure of the dimeric unit has been determined, using a novel joint refinement of intermonomer NOEs and chemical shifts. The relative position of the monomeric units in the dimer is different from that in the crystal, with less direct contact between monomers. As the temperature is raised to 70 degrees C, the peptide remains tetrameric, but the monomer units start to separate, as shown by a reduction in intermonomer NOE intensities and chemical shifts. The structural changes have been characterised: over the temperature range studied, the monomers separate by approximately 2.0 A. This movement may have implications for the mechanism by which melittin inserts into membranes.